Potent and balanced bidirectional promoter

ABSTRACT

The invention provides a bidirectional hCMV-rhCMV promoter and recombinant vectors and recombinant virus comprising the bidirectional hCMV-rhCMV promoter operably linked to a first transgene in one direction and to a second transgene in the opposite direction. The invention also provides methods of making and using such recombinant vectors and recombinant virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of parent U.S. application Ser. No.16/310,701, filed on Dec. 17, 2018, which claims priority toInternational Application Serial No. PCT/EP2017/064952, filed on Jun.19, 2017, now published as WO 2017/220499, which claims priority toEuropean Application No. 16175189.6, filed Jun. 20, 2016. The entiredisclosure of each prior application is incorporated by reference hereinin its entirety.

SEQUENCE LISTING

Pursuant to 37 C.F.R. § 1.821(c) or (e), this application contains asequence listing, which is contained on an ASCII text file entitled“Sequence Listing” (SYT 3032-CON_SequenceListing_ST25.txt, createdThursday, Mar. 4, 2021, having a size of 21,442 bytes), which is hereinincorporated by reference.

TECHNICAL FIELD

The invention relates to the field of medicine and to the field of genedelivery for applications in vaccination and gene therapy. More inparticular, the invention relates to a potent and balanced bidirectionalpromoter for the expression of two transgenes with recombinant vectors,such as plasmid vectors, viral vectors and recombinant viruses.

BACKGROUND OF THE INVENTION

Recombinant vectors are used extensively in a variety of molecularbiology applications for the expression of heterologous proteins,including, for example, their application in gene therapy andvaccination. For these gene therapy and vaccination applications,vectors, including viral vectors, are used as carriers for a gene orgenes of interest to be introduced into host cells. For example, viralvectors can be used to express a gene or part thereof encoding a desiredantigen to elicit an immune response.

The earliest viral vectors typically only included one transgene andmany strategies are published for the early generation vectors. Forexample, published strategies report the use of a variety of differentadenovirus (rAd) vectors and show that the transgene expression cassettecan be placed in different regions of the rAd, e.g., in the E1 region,the E3 region, or between E4 and the right ITR. For vaccine purposes,however, more than one antigen or the same antigen from severaldifferent strains is often required to achieve protection and broadcoverage. Therefore, in certain cases, it's desirable to express atleast two antigens from one vector. Different approaches to encode twoantigens in one viral vector have been described.

In a first two antigen approach with rAd, one antigen expressioncassette was placed in the E1 region and a second one was placed in theE3 region (e.g. (Vogels et al., 2007)). In a different two antigenapproach with rAd, one antigen expression cassette was placed in E1 anda second one between E4 and the right ITR (e.g. (Holman et al., 2007;Pham et al., 2009; Schepp-Berglind et al., 2007)). Another two antigenapproach with rAd, is to use two antigen expression cassettes placed inthe E1 region in a head-to-tail fashion using two different promotersequences in an attempt to prevent genetic instability by recombination(e.g. (Belousova et al., 2006; C. D. Harro et al., 2009)).

Another example of a two antigen approach is to use an internalribosomal entry site (IRES) of positive-stranded RNA-viruses, e.g.,derived from encephalomyocarditis virus (EMCV) to produce a singletranscript that is translated into two proteins (e.g. (Amendola,Venneri, Biffi, Vigna, & Naldini, 2005; Na & Fan, 2010)). Other examplesinclude utilizing the host cell splicing machinery or use of “cleavage”peptides derived from positive-stranded RNA viruses such as thefoot-and-mouth-disease 2A sequence or equivalents from other viruses toproduce a polyprotein that is cleaved into two proteins. According topublished reports, all of these strategies can be equally useful andsuccessful.

Alternatively, use of bidirectional promoters is another approach forexpressing two antigens with viral vectors. For example, differentbidirectional promoters have been described for lentiviral vectors(Heilbronn & Weger, 2010) and adenoviral vectors (Na & Fan, 2010; Post &Van Meir, 2001; Robbins & Ghivizzani, 1998; Walther & Stein, 2000).

In general, two different types of bidirectional promoters are known foruse, naturally occurring sequences with bidirectional properties andsynthetically designed bidirectional promoters. The naturally occurringsequences with bidirectional properties can be found in viruses, plantsor mammalian genomes (Andrianaki, Siapati, Hirata, Russell, &Vassilopoulos, 2010; Barski, Siller-Lopez, Bohren, Gabbay, &Aguilar-Cordova, 2004). For example, it has been reported that manypromoters in the human genome have some bidirectional properties. Thehuman promoters with bidirectional properties are marked by anoverrepresentation of GABP sites (Collins, Kobayashi, Nguyen, Trinklein,& Myers, 2007).

In contrast to the naturally occurring sequences, syntheticbidirectional promoters can be designed to take advantage of thedesirable properties of different unidirectional promoters. For example,Amendola et al. created two different synthetic bidirectional promotersfor use in lentiviral vectors by combining a minimal promoter derivedfrom the human cytomegalovirus (minCMV) with the human phosphoglyceratekinase promoter (PGK) or the human ubiquitin C promoter (UBI C)(Amendola et al., 2005). To construct the bidirectional promoters, theunidirectional promoters were configured in an opposite orientation(head to head), making use of only one enhancer. According to Amendolaet al., when the strong minimal promoter was combined with a fullmammalian promoter in this configuration the result was coordinateexpression from both sides. Important features for newly createdmultivalent vectors include, for example, genetic stability duringupscaling, productivity of the vector at large scale, potent expressionof both antigens, balanced expression of both antigens, and sizeconstraints of antigens expressed from the inserted expressioncassettes.

A recently described strategy that yielded particularly good resultscompared to previously disclosed methods, used a bidirectional mouseCytomegalovirus (mCMV) promoter to express two transgenes (WO2016/166088). Therein, a first transgene was operably linked to thebidirectional mCMV promoter in one direction and a second transgene wasoperably linked to the bidirectional mCMV promoter in the otherdirection. The rAd with the bidirectional mCMV promoter were determinedto be genetically stable, providing genetic stability that wascomparable to rAd with only a single transgene. Furthermore, it wasdetermined that both transgenes were sufficiently expressed to generateimmunogenic responses to both antigens based on ELISPOT and ELISAanalysis of the immunogenicity of the expressed antigens with regard toT-cell and B-cell responses. The mCMV bidirectional promoter was thusdescribed to be superior to several other previously describedstrategies. However, it was determined that the balance of theexpression levels between both sides of the mCMV promoter could befurther improved. There was approximately a 10-times higher expressionof an antigen positioned at the right side (3′ end) of the bidirectionalmCMV promoter compared to the antigen positioned at the left side (5′end) of the promoter. The imbalance in expression of the two encodedantigens leads to a stronger immune response directed against the highlyexpressed antigen compared to the lower expressed antigen. This kind ofdifferential expression could be useful for certain applications, butfor other applications it is also desirable to have a strategy thatcombines several advantages of the mCMV promoter with a more balancedexpression, i.e. a bidirectional promoter that is both potent and morebalanced than the bidirectional mCMV promoter and other bidirectionalpromoters that have been described in the literature.

Thus, a need remains to identify bidirectional promoters that arepotent, relatively short, have no or limited lengthy internal stretchesof identical sequences, and have an improved balance in expression fromboth sides compared to the mCMV bidirectional promoter, and to providerecombinant viruses that are genetically stable with potent and balancedexpression of two transgenes.

SUMMARY OF THE INVENTION

The present invention provides recombinant nucleic acid moleculescomprising a bidirectional hCMV-rhCMV promoter and vectors, including,for example, plasmid vectors, viral vectors, and viruses comprising thebidirectional hCMV-rhCMV promoter. The recombinant vectors of thepresent invention comprise two transgenes, wherein the transcriptionaldirection (5′ to 3′) of the hCMV and rhCMV portions of the hCMV-rhCMVbidirectional promoter point away from each other (head to headconfiguration), wherein a first transgene is operably linked in onedirection on the left side, with expression controlled by the hCMVportion of the bidirectional promoter, and a second transgene isoperably linked in the opposite direction on the right side, withexpression controlled by the rhCMV portion of the bidirectionalpromoter. The hCMV enhancer is placed in the middle between the twodifferent promoters pointing towards the hCMV promoter part. Sinceenhancers can be orientation-independent the enhancer providescoordinate expression of both transgenes operably linked to the hCMV andthe rhCMV portions of the bidirectional promoter. See, for example, FIG.1D shows the identity and orientation for different building blocks of arepresentative hCMV-rhCMV promoter. Preferably, a hCMV-rhCMV promoteraccording to the invention comprises a nucleotide sequence that is atleast 80%, preferably at least 85%, more preferably at least 90%, stillmore preferably at least 95%, and up to 100%, identical to SEQ ID NO: 4.

In certain embodiments, the recombinant viruses and recombinant viralvectors are recombinant adenoviruses (rAd) and rAd vectors. The rAdproduced with the bidirectional hCMV-rhCMV promoter of the presentinvention are genetically stable, with no deletion bands detected by PCRanalysis up to passage 13 (p13), thus providing genetic stability thatis comparable to viruses with only a single transgene. Furthermore, thebidirectional hCMV-rhCMV promoter is a relatively short bidirectionalpromoter with only 943 nucleotides, and it provides potent and verybalanced expression of the two transgenes. Thus, the bidirectionalhCMV-rhCMV promoter of the present invention is suitable for use in genetherapy and vaccine applications with recombinant (viral) vectors, andin particular where very balanced and potent expression are importantand/or where the small size of the bidirectional hCMV-rhCMV promoter isuseful.

The general and preferred embodiments are defined, respectively, by theindependent and dependent claims appended hereto, which for the sake ofbrevity are incorporated by reference herein. Other preferredembodiments, features, and advantages of the various aspects of theinvention will become apparent from the detailed description below takenin conjunction with the appended drawing figures.

In one embodiment, the present invention provides a bidirectionalhCMV-rhCMV promoter comprising the hCMV promoter on the left side andthe rhCMV promoter on the right side, wherein the bidirectionalhCMV-rhCMV promoter is operably linked to a first transgene in onedirection on the left side and the bidirectional hCMV-rhCMV promoter isoperably linked to a second transgene on the right side in the otherdirection.

In another embodiment, the present invention also provides a method ofproducing a recombinant virus comprising a first and a second transgene,the method comprising: preparing a construct comprising a bidirectionalhCMV-rhCMV promoter operably linked to a first transgene in onedirection and to a second transgene in the opposite direction, andincorporating said construct into the genome of the recombinant virus.

In certain embodiments, the recombinant virus is a recombinantadenovirus.

In certain embodiments, the recombinant adenovirus has a deletion in theE1 region, and in certain embodiments comprises the bidirectionalhCMV-rhCMV promoter and first and second transgene in this E1 region.Alternatively, other regions of the recombinant adenovirus could also beused. For example, the bidirectional promoter expression cassette couldalso be placed at the right end of the genome, between the E4 region andthe right ITR of the recombinant adenovirus.

In certain embodiments, the first and second transgene are different andat least one of them encodes an antigen. In certain embodiments bothencode a different antigen.

In certain embodiments, the adenovirus is a human adenovirus serotype 26or a human adenovirus serotype 35.

In another embodiment, the present invention also provides a method forexpressing at least two transgenes in a cell, the method comprisingproviding a cell with a recombinant vector according to the invention.

In another embodiment, the present invention also provides a method forinducing an immune response against at least two antigens, the methodcomprising administering to a subject a recombinant vector according tothe invention.

In another embodiment, the present invention also provides a recombinantDNA molecule comprising the genome of a recombinant adenovirus accordingto the invention.

In another embodiment, the present invention also provides apharmaceutical composition comprising a recombinant vector, such as arecombinant adenovirus, according to the invention and apharmaceutically acceptable carrier or excipient. In certainembodiments, the pharmaceutical composition is a vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1J: Schematic representations of tested bidirectionalpromoter constructs including annotations for the identity andorientation of building blocks for the different bidirectional promotersequences. P: promoter, Enh: enhancer, I: intron.

FIGS. 2A to 2C: Expression of Luciferase and eGFP with differentbidirectional promoter constructs evaluated with transient transfectionsin HEK293 cells. Luciferase expression is measured as relative lightunits (RLU) and eGFP expression is measured as mean fluorescenceintensity (MFI) by FACS. Results of three different experimentsscreening different promoter constructs are shown. Shown are bar graphsof the results for Luciferase expression from the left side and eGFPexpression from the right side of different bidirectional promoters.Positive control: Luciferase or eGFP under control of a unidirectionalhCMV promoter; untransfected cells are used as a negative control.

FIGS. 3A and 3B: (A) Organization of bidirectional expression cassettefor bidirectional promoter hCMV-rhCMV in pshuttle26, including theidentity and locations for restriction sites used to insert transgeneson both sides of the bidirectional promoter construct. P: promoter, Enh:enhancer, TG: transgene, pA: polyadenylation signal, derived from SV40(right side) or bovine growth hormone (BGH) (left side). Representationin plasmid vector pshuttle26. The same bidirectional expression cassetteorganization was used in pAdapt35. (B) Schematic representation ofhCMV-rhCMV bidirectional promoter including the nucleotide positions ofthe building blocks. The arrows represent the direction oftranscription.

FIGS. 4A and 4B: Expression of Luciferase and eGFP transgenes on eitherthe left or right side of bidirectional promoter constructs in Ad26 rAdvectors (A) and Ad35 rAd vectors (B) with infections in A549 cells at1000 VP/cell. Luciferase expression is measured as relative light units(RLU) and eGFP expression is measured as mean fluorescence intensity(MFI) by FACS. Results for the different hCMV-rhCMV bidirectionalpromoter constructs are compared to positive controls of 100 VP/cell and1000 VP/cell for Luciferase or eGFP under control of a unidirectionalhCMV promoter and to cells infected with an empty vector. For Ad26 rAdvectors, hCMV-rhCMV is additionally compared to the bidirectional mCMVpromoter.

FIG. 5: Genetic stability testing by serial propagation followed by PCRon Ad26 vector genome harboring the bidirectional hCMV-rhCMV promoter inthe E1 region and encoding eGFP and Luciferase on either the right orleft side of the bidirectional hCMV-rhCMV promoter. Shown in the panelsfrom top to bottom are PCR products for 5 plaques per vector afterserial propagation in PER.C6 cells at P5, P10, and P13. Lanes 1-5 ineach panel show the bidirectional hCMV-rhCMV promoter with Luciferase onthe left and eGFP on the right. Lanes 6-10 in each panel show thebidirectional hCMV-rhCMV promoter with eGFP on the left and luciferaseon the right. Lane 11 shows the kB marker. Lane 12 shows the plasmidpositive control for Ad26.Luc.hCMV-rhCMV.eGFP. Lane 13 shows the plasmidpositive control for rAd26.eGFP.hCMV-rhCMV.Luc. Lane 14 shows theplasmid control of the PCR product size of an expression cassettewithout transgene. Lane 15 shows the negative water PCR control.Labelling: P5, P10, P13: viral passage number. Additional bands besidesthe expected PCR products are unspecific PCR products. Note: Absence ofdeletion bands was confirmed on overexposed pictures.

DETAILED DESCRIPTION OF THE INVENTION

Described herein are experimental results comparing new bidirectionalpromoter constructs for potency and balance. The results show that thebidirectional hCMV-rhCMV promoter provides potent and very balancedexpression of two transgenes, based on transient transfection withpAdApt plasmid vectors in HEK293 cells and viral infections with rAd26and rAd35 comprising the bidirectional hCMV-rhCMV promoter with a firsttransgene operably linked to the bidirectional hCMV-rhCMV promoter inone direction and a second transgene operably linked to thebidirectional hCMV-rhCMV promoter in the other direction. Thebidirectional hCMV-rhCMV promoter is also a relatively shortbidirectional promoter with only 943 nucleotides. Furthermore, rAd withthe bidirectional hCMV-rhCMV promoter are genetically stable, with nodeletion bands detected by PCR analysis up to passage 13 (p13), thusproviding genetic stability that is comparable to viruses with only asingle transgene. Thus, the rAd of the present invention with thebidirectional hCMV-rhCMV promoter are suitable for use in gene therapyand vaccine applications where very balanced and potent expression are apriority and/or where the small size of the bidirectional hCMV-rhCMVpromoter is useful, e.g. to leave more space for transgenes in thelimited size of the vector or viral genome, as compared to other, longerbidirectional promoters.

The present invention therefore relates to recombinant nucleic acidmolecules comprising a bidirectional hCMV-rhCMV promoter operably linkedto a first transgene in one direction and to a second transgene in theopposite direction, wherein the transcriptional direction (5′ to 3′) ofthe hCMV and rhCMV portions of the hCMV-rhCMV bidirectional promoterpoint away from each other, and wherein expression from the left side iscontrolled by the hCMV portion of the bidirectional promoter andexpression from the right side is controlled by the rhCMV portion of thebidirectional promoter. In certain embodiments, the invention relates tousing vectors, viral vectors, and viruses comprising the bidirectionalhCMV-rhCMV promoter for expressing two transgenes in a cell.

In certain embodiments, the invention relates to plasmid vectors for usein enabling host cells to produce heterologous proteins. For example,plasmid vectors comprising the bidirectional hCMV-rhCMV promoter couldbe used for expressing two different components of a heteromericmulti-subunit protein complex. Such plasmid vectors could be DNAsequences containing, for example, (1) the bidirectional hCMV-rhCMVpromoter; (2) sequences providing mRNA with a ribosome binding site foreach transgene; (3) a coding region for each transgene, i.e., a sequenceof nucleotides which codes for the desired polypeptide; (4) a Kozakconsensus sequence for each transgene for initiation of translation; (5)a termination sequence for each transgene which permits translation tobe terminated when the entire code for each transgene has been read; and(6) if the vector is not directly inserted into the genome, an origin ofreplication which permits the entire vector to be reproduced once it iswithin the cell. It then remains to induce the host cell to incorporatethe vector, for example by transfection or electroporation, and to growthe host cells in such a way as to express the two transgenes as part ofthe host cell's function.

In certain embodiments, the invention relates to rAd and rAd vectorscomprising the bidirectional hCMV-rhCMV promoter and methods of makingand using the rAd and rAd vectors, wherein the rAd and rAd vectorscomprise a bidirectional hCMV-rhCMV promoter and two transgenes, whereina first transgene is operably linked to the bidirectional hCMV-rhCMVpromoter in one direction and a second transgene is operably linked tothe bidirectional hCMV-rhCMV promoter in the other direction.

The rAd of the present invention can be produced in large amounts, orbatches. A ‘batch’ of rAd is a composition that has been produced in oneproduction run in a single production vessel, or alternatively it canrefer to the plurality of rAd particles in a composition that is presentin a single container (e.g., bioreactor, bag, flask, bottle, multi-dosevial, single-dose vial, syringe, etc). A batch of rAd according to theinvention or a composition comprising rAd according to the inventionpreferably comprises at least 10⁷ rAd particles, and in certainembodiments comprises at least 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴,10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, or more rAd particles, up to 10²⁰ rAd particles(e.g. as produced in a large scale bioreactor in a single productionrun). A batch or composition may or may not comprise further relevantcomponents besides the rAd.

The term ‘recombinant’ for a recombinant adenovirus, as used hereinimplicates that it has been modified by the hand of man as opposed towild-type adenoviruses, e.g. it comprises a heterologous gene, genes, orparts thereof and a bidirectional hCMV-rhCMV promoter.

Sequences herein are provided in the 5′ to 3′ direction, as is customaryin the art.

An “adenovirus capsid protein” refers to a protein on the capsid of anadenovirus that is involved in determining the serotype and/or tropismof a particular adenovirus. Adenoviral capsid proteins typically includethe fiber, penton and/or hexon proteins. A rAd of (or ‘based upon’) acertain serotype according to the invention typically comprises fiber,penton and/or hexon proteins of that certain serotype, and preferablycomprises fiber, penton and hexon protein of that certain serotype.These proteins are typically encoded by the genome of the rAd. A rAd ofa certain serotype may optionally comprise and/or encode other proteinsfrom other adenovirus serotypes.

A rAd is ‘based upon’ an adenovirus as used herein, by derivation fromthe wild type, at least in sequence. This can be accomplished bymolecular cloning, using the wild type genome or parts thereof asstarting material. It is also possible to use the known sequence of awild type adenovirus genome to generate (parts of) the genome de novo byDNA synthesis, which can be performed using routine procedures byservice companies having business in the field of DNA synthesis and/ormolecular cloning (e.g. GeneArt, GenScripts, Invitrogen, Eurofins).Thus, as a non-limiting example, a rAd that comprises hexon, penton andfiber of Ad35 is considered a rAd based upon Ad35, etc.

The adenoviral vectors of the present invention are referred to as rAdvectors. The preparation of rAd vectors is well known in the art.

In certain embodiments, a rAd vector according to the invention isdeficient in at least one essential gene function of the E1 region, e.g.the Ela region and/or the E1b region, of the adenoviral genome that isrequired for viral replication. In certain embodiments, an adenoviralvector according to the invention is deficient in at least part of thenon-essential E3 region. In certain embodiments, the vector is deficientin at least one essential gene function of the E1 region and at leastpart of the non-essential E3 region. The adenoviral vector can be“multiply deficient,” meaning that the adenoviral vector is deficient inone or more essential gene functions in each of two or more regions ofthe adenoviral genome. For example, the aforementioned E1-deficient orE1-, E3-deficient adenoviral vectors can be further deficient in atleast one essential gene of the E4 region and/or at least one essentialgene of the E2 region (e.g., the E2A region and/or E2B region).

Adenoviral vectors, methods for construction thereof and methods forpropagating thereof, are well known in the art and are described in, forexample, U.S. Pat. Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806,5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191,6,113,913, and 8,932,607, and Thomas Shenk, “Adenoviridae and theirReplication” M. S. Horowitz, “Adenoviruses”, Chapters 67 and 68,respectively, in Virology, B. N. Fields et al., eds., 3d ed., RavenPress, Ltd., New York (1996), and other references mentioned herein.Typically, construction of adenoviral vectors involves the use ofstandard molecular biological techniques that are well known in the art,such as those described in, for example, Sambrook et al., MolecularCloning, a Laboratory Manual, 2d ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (1989), Watson et al., Recombinant DNA, 2d ed.,Scientific American Books (1992), and Ausubel et al., Current Protocolsin Molecular Biology, Wiley Interscience Publishers, NY (1995), andother references mentioned herein.

An adenovirus according to the invention belongs to the family of theAdenoviridae and preferably is one that belongs to the genusMastadenovirus. It can be a human adenovirus, but also an adenovirusthat infects other species, including but not limited to a bovineadenovirus (e.g. bovine adenovirus 3, BAdV3), a canine adenovirus (e.g.CAdV2), a porcine adenovirus (e.g. PAdV3 or 5), or a simian adenovirus(which includes a monkey adenovirus and an ape adenovirus, such as achimpanzee adenovirus or a gorilla adenovirus). Preferably, theadenovirus is a human adenovirus (HAdV, or AdHu; in the presentinvention a human adenovirus is meant if referred to Ad withoutindication of species, e.g. the brief notation “Ads” means the same asHAdV5, which is human adenovirus serotype 5), or a simian adenovirussuch as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV).

Most advanced studies have been performed using human adenoviruses, andhuman adenoviruses are preferred according to certain aspects of theinvention. In certain preferred embodiments, the recombinant adenovirusaccording to the invention is based upon a human adenovirus. Inpreferred embodiments, the recombinant adenovirus is based upon a humanadenovirus serotype 5, 11, 26, 34, 35, 48, 49 or 50. According to aparticularly preferred embodiment of the invention, an adenovirus is ahuman adenovirus of one of the serotypes 26 and 35. An advantage ofthese serotypes is a low seroprevalence and/or low pre-existingneutralizing antibody titers in the human population. Preparation ofrAd26 vectors is described, for example, in WO 2007/104792 and in(Abbink et al., 2007). Exemplary genome sequences of Ad26 are found inGenBank Accession EF 153474 and in SEQ ID NO:1 of WO 2007/104792.Preparation of rAd35 vectors is described, for example, in U.S. Pat. No.7,270,811, in WO 00/70071, and in (Vogels et al., 2003). Exemplarygenome sequences of Ad35 are found in GenBank Accession AC_000019 and inFIG. 6 of WO 00/70071.

Simian adenoviruses generally also have a low seroprevalence and/or lowpre-existing neutralizing antibody titers in the human population, and asignificant amount of work has been reported using chimpanzee adenovirusvectors (e.g. U.S. Pat. No. 6,083,716; and WO 2005/071093; WO2010/086189; and WO 2010085984; (Bangari & Mittal, 2006; Cohen et al.,2002; Farina et al., 2001; Kobinger et al., 2006; Lasaro & Ertl, 2009;Tatsis et al., 2007). Hence, in other preferred embodiments, therecombinant adenovirus according to the invention is based upon a simianadenovirus, e.g. a chimpanzee adenovirus. In certain embodiments, therecombinant adenovirus is based upon simian adenovirus type 1, 7, 8, 21,22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36,37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 or SA7P. Alsorhesus monkey adenovirus vectors have been described as useful candidatevectors (e.g. (Abbink et al., 2015); WO 2014/078688). Hence, in otherpreferred embodiments, the recombinant adenovirus of the invention isbased upon a rhesus monkey adenovirus, for instance on one of thenon-limiting examples RhAd51, RhAd52 or RhAd53 (or sAd4287, sAd4310A orsAd4312; see e.g. (Abbink et al., 2015) and WO 2014/078688).

In addition to adenoviruses, those skilled in the art will recognizethat other viruses are also suitable for use as viral vectors using thebidirectional promoters of the present invention. For example,adeno-associated viruses (AAV), herpes simplex virus (HSV), poxvirus andlentivirus can also be engineered to include the bidirectional promotersof the present invention. See, for example, reviews about differentvectors as discussed in (Heilbronn & Weger, 2010; Robbins & Ghivizzani,1998; Walther & Stein, 2000).

The sequences of most of the human and non-human adenoviruses mentionedabove are known, and for others can be obtained using routineprocedures.

A recombinant adenovirus according to the invention may bereplication-competent or replication-deficient.

In certain embodiments, the adenovirus is replication deficient, e.g.because it contains a deletion in the E1 region of the genome. A“deletion in the E1 region” means a deletion in this region as comparedto a wild-type adenovirus, and means a deletion in at least one of theE1A, E1B 55K or E1B 21K coding regions, preferably a deletion of E1A,E1B 55K and E1B21K coding regions. As known to the skilled person, incase of deletions of essential regions from the adenovirus genome, thefunctions encoded by these regions have to be provided in trans,preferably by the producer cell, i.e. when parts or whole of E1, E2and/or E4 regions are deleted from the adenovirus, these have to bepresent in the producer cell, for instance integrated in the genomethereof, or in the form of so-called helper adenovirus or helperplasmids. The adenovirus may also have a deletion in the E3 region,which is dispensable for replication, and hence such a deletion does nothave to be complemented.

A producer cell (sometimes also referred to in the art and herein as‘packaging cell’ or ‘complementing cell’ or ‘host cell’) that can beused can be any producer cell wherein a desired adenovirus can bepropagated. For example, the propagation of recombinant adenovirusvectors is done in producer cells that complement deficiencies in theadenovirus. Such producer cells preferably have in their genome at leastan adenovirus E1 sequence, and thereby are capable of complementingrecombinant adenoviruses with a deletion in the E1 region. AnyE1-complementing producer cell can be used, such as human retina cellsimmortalized by E1, e.g. 911 or PER.C6 cells (see, e.g., U.S. Pat. No.5,994,128), E1-transformed amniocytes (See, e.g., EP 1230354),E1-transformed A549 cells (see e.g. WO 98/39411, U.S. Pat. No.5,891,690), GH329:HeLa cells (Gao, Engdahl, & Wilson, 2000), 293 cells,and the like. In certain embodiments, the producer cells are forinstance HEK293 cells, or PER.C6 cells, or 911 cells, or IT293SF cells,and the like.

For E1-deficient adenoviruses that are not derived from subgroup C or Eadenoviruses, it is preferred to exchange the E4-orf6 coding sequence ofthe non-subgroup C or E adenovirus with the E4-orf6 of an adenovirus ofsubgroup C such as Ad5. This allows propagation of such adenoviruses inwell-known complementing cell lines that express the E1 genes of Ad5,such as for example 293 cells or PER.C6 cells (see, e.g. (Havenga etal., 2006); WO 03/104467, incorporated in its entirety by referenceherein).

In alternative embodiments, there is no need to place a heterologousE4orf6 region (e.g. of Ad5) in the adenoviral vector, but instead theE1-deficient non-subgroup C or E vector is propagated in a cell linethat expresses both E1 and a compatible E4orf6, e.g. the 293-ORF6 cellline that expresses both E1 and E4orf6 from Ad5 (see e.g. (Brough,Lizonova, Hsu, Kulesa, & Kovesdi, 1996) describing the generation of the293-ORF6 cells; (Abrahamsen et al., 1997; Nan et al., 2003) eachdescribing generation of E1 deleted non-subgroup C adenoviral vectorsusing such a cell line).

Alternatively, a complementing cell that expresses E1 from the serotypethat is to be propagated can be used (see e.g. WO 00/70071, WO02/40665).

For subgroup B adenoviruses, such as Ad35, having a deletion in the E1region, it is preferred to retain the 3′ end of the MB 55K open readingframe in the adenovirus, for instance the 166 bp directly upstream ofthe pIX open reading frame or a fragment comprising this such as a 243bp fragment directly upstream of the pIX start codon (marked at the 5′end by a Bsu36I restriction site in the Ad35 genome), since thisincreases the stability of the adenovirus because the promoter of thepIX gene is partly residing in this area (see, e.g. (Havenga et al.,2006); WO 2004/001032, incorporated by reference herein).

“Heterologous nucleic acid” (also referred to herein as ‘transgene’) invectors or (adeno)viruses of the invention is nucleic acid that is notnaturally present in the vector or (adeno)virus. It is introduced intothe vector or (adeno)virus for instance by standard molecular biologytechniques. It may in certain embodiments encode a protein of interestor part thereof. It can for instance be cloned into a deleted E1 or E3region of an adenoviral vector. In preferred embodiments of theinvention, the expression cassette with the two transgenes under controlof the bidirectional hCMV-rhCMV promoter is placed into the E1 region ofthe adenoviral genome. A transgene is generally operably linked toexpression control sequences. This can for instance be done by placingthe nucleic acid encoding the transgene(s) under the control of apromoter. Many promoters can be used for expression of a transgene(s),and are known to the skilled person.

It is known that homologous stretches of nucleic acid could lead toinstability. For example, using two identical (hCMV) promoters in oneadenovirus vector, while previously reported to be possible, upon moreextensive testing appeared to lead to genetic instability of theadenovirus (WO 2016/166088). The present inventors therefore tried tominimize using promoter building blocks having extensive stretches ofsequence identity when designing bidirectional promoters, in order toprevent deletions by homologous recombination in the adenoviral vector.Importantly, adenovirus vectors with transgenes regulated by thehCMV-rhCMV bidirectional promoter of the invention were shown to begenetically stable herein.

As used herein, the terms “promoter” or “promoter region” or “promoterelement” are used interchangeably, and refer to a segment of a nucleicacid sequence, typically but not limited to DNA, that controls thetranscription of the nucleic acid sequence to which it is operativelylinked. The promoter region includes specific sequences that aresufficient for RNA polymerase recognition, binding and transcriptioninitiation. In addition, the promoter region can optionally includesequences which modulate this recognition, binding and transcriptioninitiation activity of RNA polymerase. These sequences may be cis-actingor may be responsive to trans-acting factors. Furthermore, the promotersmay be constitutive or regulated, depending upon the nature of theregulation.

The skilled person will be aware that promoters are built from stretchesof nucleic acid sequences and often comprise elements or functionalunits in those stretches of nucleic acid sequences, such as atranscription start site, a binding site for RNA polymerase, generaltranscription factor binding sites, such as a TATA box, specifictranscription factor binding sites, and the like. Further regulatorysequences may be present as well, such as enhancers, and sometimesintrons at the end of a promoter sequence. Such functional units arereferred to herein below as ‘building blocks’, and they may be combinedin a stretch of nucleic acid to build a functional promoter sequence.The building blocks may be directly adjacent to each other but may alsobe separated by stretches of nucleic acid that do not have a direct rolein the promoter function. The skilled person knows how to test whethernucleotides in the stretch of nucleic acid are relevant for promoterfunction, and to how to remove or add building blocks and/or nucleotidesinto a given promoter sequence by standard molecular biology methods,e.g. to minimize its length while retaining promoter activity or tooptimize activity.

As used herein, the terms “enhancer” or “enhancer building block” referto regulatory DNA sequences, e.g., 50-1500 bp, that can be bound byproteins (activator proteins) to stimulate or enhance transcription of agene or several genes. These activator proteins, (a.k.a., transcriptionfactors) interact with the mediator complex and recruit polymerase IIand the general transcription factors which then begin transcribing thegenes. Enhancers are generally cis-acting, but can be located eitherupstream or downstream from the start site of the gene or genes theyregulate. Furthermore, an enhancer can be either in the forward orbackward direction and doesn't need to be located near the transcriptioninitiation site to affect transcription, as some have been found locatedseveral hundred thousand base pairs upstream or downstream of the startsite. Enhancers can also be found within introns.

The term “bidirectional promoter” refers to continuous gene regulatorysequences that may contain enhancer elements and intron elements besidesthe promoter elements and are defined by the building blocks asdescribed herein. These bidirectional promoters direct gene expressionin a bidirectional fashion controlling expression for transgenes placedon both sides of the bidirectional promoter sequence. For example, thebidirectional promoter of the present invention directs transcription oftwo different transgenes in a bidirectional fashion and includes anenhancer building block flanked by a first promoter building block onone side and a second promoter building block on the other side, suchthat the transgenes are downstream of the respective promoter buildingblocks. Note that flanked and adjacent do not necessarily mean directlycontiguous as there might be some additional nucleotides in between thebuilding blocks, but preferably not too much additional sequence isadded so that the bidirectional promoter maintains a compact size. Alsonote that the terms ‘upstream’ and ‘downstream’ are with respect to thedirection of transcription as commonly used in the art. For example, byconvention the terms upstream and downstream relate to the 5′ to 3′direction in which RNA transcription takes place. Upstream is toward the5′ end of the RNA molecule and downstream is toward the 3′ end. Whenconsidering double-stranded DNA, upstream is toward the 5′ end of thecoding strand for the gene in question and downstream is toward the 3′end. Due to the anti-parallel nature of DNA, this means the 3′ end ofthe template strand is upstream of the gene and the 5′ end isdownstream. See, for example, FIG. 1D, shows a preferred bidirectionalhCMV-rhCMV promoter comprising a human cytomegalovirus major immediateearly enhancer (hCMV enhancer) as an enhancer building block flanked bya human cytomegalovirus major immediate early promoter (hCMV) as a firstpromoter building block on one side and a rhesus CMV promoter (rhCMV) asa second promoter building block on the other side. The bidirectionalhCMV-rhCMV promoter of the present invention is operably linked to twotransgenes, such that a first transgene is operably linked to the hCMVpromoter building block, and a second transgene is operably linked tothe rhCMV promoter building block, such that the first and secondtransgenes each are located downstream of the respective promoter andsuch that the first and second transgenes are transcribed in a directionoutward from the hCMV enhancer.

A preferred bidirectional promoter of the present invention is thebidirectional hCMV-rhCMV promoter comprising SEQ ID NO:4, with thesequence locations for the different elements as indicated in FIG. 3B,but a person skilled in the art will recognize that the length of oridentity in the sequences of the different building blocks and theintervening sequences could be varied to some degree such thatessentially similar results could be obtained. For example, differentenhancers could be tested for substitution and/or the enhancer sequencescould be tweaked such that essentially similar expression could beobtained. Similarly, an intron could be added adjacent to and downstreamof one or both of the promoter building blocks and it is expected thatthe bidirectional hCMV-rhCMV promoter would still be active. Possibly,this could even lead to enhanced expression, but in any case this couldgo at the expense of the space for the transgene as the intron(s) wouldtake up space in the vector or virus. The enhancers indicated herein arepreferred, being of suitable sizes, giving rise to balanced expression,and stable constructs in an adenoviral vector context. The buildingblocks of the bidirectional promoter of the invention as such may havebeen individually known, but were never combined nor even suggested tobe combined in the constellation of the invention, which results in apotent, very balanced and relatively short bidirectional promoter. Asshown herein, despite having a relatively short sequence of only 943nucleotides, this novel bidirectional combination of the hCMV enhancerwith the hCMV and rhCMV promoters was surprisingly found to be capableof directing potent and balanced transcription of two operably linkedtransgenes, while at the same time remaining a stable configuration ofthe bidirectional promoter with associated transgenes in the complexcontext of an adenoviral vector. Data presented herein show thatcreating such bidirectional promoters based upon known similar buildingblocks was unpredictable, in that several other similarly designedbidirectional promoters either lacked strong promoter activity, and/orled to unbalanced expression whereby expression of the transgeneoperably linked to one part of a bidirectional promoter was expressedsignificantly stronger (e.g. at least 5× difference) compared to thetransgene operably linked to the other part of such a bidirectionalpromoter. It was a priori not predictable whether any promoter thatwould meet the requirements of similar expression levels from both sides(e.g. less than 2× difference between expression from both sides) andstability in the context of adenoviral vectors, would be achievable atall. The present invention surprisingly provides bidirectional promotersthat meet these requirements, and have a small size, which can be highlyadvantageous in the context of size limitations of vectors carryingtransgenes (i.e. larger transgenes can be accommodated and/or thevectors could remain more stable).

Further regulatory sequences may also be added to constructs comprisingthe bidirectional promoters of the present invention. The term“regulatory sequence” is used interchangeably with “regulatory element”herein and refers to a segment of nucleic acid, typically but notlimited to DNA, that modulate the transcription of the nucleic acidsequence to which it is operatively linked, and thus acts as atranscriptional modulator. A regulatory sequence often comprises nucleicacid sequences that are transcription binding domains that arerecognized by the nucleic acid-binding domains of transcriptionalproteins and/or transcription factors, enhancers or repressors etc. Forexample, a regulatory sequence could include one or more tetracyclineoperon operator sequences (tetO), such that expression is inhibited inthe presence of the tetracycline operon repressor protein (tetR). In theabsence of tetracycline, the tetR protein is able to bind to the tetOsites and repress transcription of a gene operably linked to the tetOsites. In the presence of tetracycline, however, a conformational changein the tetR protein prevents it from binding to the operator sequences,allowing transcription of operably linked genes to occur. In certainembodiments, rAd of the present invention can optionally include tetOoperatively linked to the bidirectional hCMV-rhCMV promoter, such thatexpression of one or more transgenes is inhibited in the vectors thatare produced in the producer cell line in which tetR protein isexpressed. Subsequently, expression would not be inhibited if the vectoris introduced into a subject or into cells that do not express the tetRprotein (see e.g., WO 07/073513). In certain other embodiments, vectorof the present invention can optionally include a cumate gene-switchsystem, in which regulation of expression is mediated by the binding ofthe repressor (CymR) to the operator site (CuO), placed downstream ofthe promoter (see e.g., (Mullick et al., 2006)).

As used herein, the term “repressor,” refers to entities (e.g., proteinsor other molecules) having the capacity to inhibit, interfere, retardand/or repress the production of heterologous protein product of arecombinant expression vector. For example, by interfering with abinding site at an appropriate location along the expression vector,such as in an expression cassette. Examples of repressors include tetR,CymR, the lac repressor, the trp repressor, the gal repressor, thelambda repressor, and other appropriate repressors known in the art.

Furthermore, a recombinant vector, virus or adenovirus of the presentinvention comprises a bidirectional hCMV-rhCMV promoter, wherein thetranscriptional direction (5′ to 3′) of the hCMV and rhCMV portions ofthe hCMV-rhCMV bidirectional promoter point away from each other, andwherein the hCMV-rhCMV bidirectional promoter is operably linked to afirst transgene in one direction and to a second transgene in theopposite direction. The bidirectional promoter thus will driveexpression of the first transgene towards a first end of the vector or(adeno)viral genome and of the second transgene towards the other end ofthe vector or (adeno)viral genome. The skilled person will be aware thatmutations can be made in the provided sequences and can be tested forpromoter activity by routine methods. Typically, a sequence having atleast 90% identity with the indicated promoter sequences (not includingthe enhancer sequences) will still have functional activity and hencewill be considered a bidirectional hCMV-rhCMV promoter. Thus, thebidirectional hCMV-rhCMV promoter of the present invention preferablyhas at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity to the indicated promoter sequences (outside the enhancersequence). Preferably, the bidirectional hCMV-rhCMV promoter comprises asequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto SEQ ID NO: 4. In certain preferred embodiments, the bidirectionalhCMV-rhCMV promoter contains the building blocks as shown in FIG. 1D,wherein the bidirectional hCMV-rhCMV promoter comprises an hCMV enhanceras an enhancer building block flanked by an hCMV promoter as a firstpromoter building block on one side and a rhesus CMV (rhCMV) promoter asa second promoter building block on the other side. In a certain otherpreferred embodiment the bidirectional hCMV-rhCMV promoter is 100%identical to SEQ ID NO:4, with the sequence locations for the differentelements as indicated in FIG. 3, but a person skilled in the art willrecognize that the length of the sequences of the different buildingblocks and the intervening sequences could be varied to some degree andthe identity of the enhancer could also be varied such that essentiallysimilar results could be obtained.

The terms “operably linked”, or “operatively linked” are usedinterchangeably herein, and refer to the functional relationship of thenucleic acid sequences with regulatory sequences of nucleotides, such aspromoters, enhancers, transcriptional and translational stop sites, andother signal sequences and indicates that two or more DNA segments arejoined together such that they function in concert for their intendedpurposes. For example, operative linkage of nucleic acid sequences,typically DNA, to a regulatory sequence or promoter region refers to thephysical and functional relationship between the DNA and the regulatorysequence or promoter such that the transcription of such DNA isinitiated from the regulatory sequence or promoter, by an RNA polymerasethat specifically recognizes, binds and transcribes the DNA. In order tooptimize expression and/or in vitro transcription, it may be necessaryto modify the regulatory sequence for the expression of the nucleic acidor DNA in the cell type for which it is expressed. The desirability of,or need of, such modification may be empirically determined.

The expression controlled by either part of the bidirectional hCMV-rhCMVpromoter the transgene is potently expressed. As used herein, “potentlyexpressed” or “potent expression” mean that the expression from eitherpart of the bidirectional hCMV-rhCMV promoter, as measured for exampleby different protein detection techniques such as Western Blot, FACSanalysis, or other assays using luminescence or fluorescence, is atleast 10%, preferably at least 20%, more preferably at least 30% ofexpression from a monovalent vector expressing a single transgene underthe control of a unidirectional hCMV promoter (having SEQ ID NO: 9). Ofnote, the unidirectional hCMV promoter is much stronger compared toother commonly used unidirectional promoters such as PGK, UBI C or RSVLTR promoters (Powell, Rivera-Soto, & Gray, 2015). Therefore, abidirectional promoter which is less strong than the hCMV promoter (e.g.leading to an expression level that is at least 10% of such aunidirectional hCMV promoter) can still be considered potent. The hCMVpromoters are derived from the major immediate early (mIE) region ofhuman cytomegalovirus and are frequently used for potent unidirectionalgene expression in vaccine and gene therapy vectors. For example, a hCMVpromoter sequence can be derived from the hCMV AD169 strain mIE locus(X03922) and include NF1 binding sites, the enhancer region, TATA boxand part of the first exon. Other hCMV promoter sequences are knownwhich can be shorter (e.g. only containing the enhancer and promoterregion and lacking NF1 binding sites) or longer (e.g. includingadditional cellular factor binding sites and the first intron sequence).These hCMV promoters which differ in length were all found to be potentubiquitously active promoters. For the comparisons of expression levelsas described herein, the hCMV promoter sequence was SEQ ID NO:9. Forexample, the expression level from either part of the bidirectionalhCMV-rhCMV promoter of the present invention in a rAd is preferably atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of theexpression level from a rAd where the transgene is under control of aunidirectional hCMV promoter of SEQ ID NO:9. In certain embodiments, theexpression level from either part of the bidirectional hCMV-rhCMVpromoter is about 10-60%, e.g. 20-50%, e.g. about 30%, of the expressionlevel from a rAd where the transgene is under control of aunidirectional hCMV promoter of SEQ ID NO:9. Furthermore, it is knownfrom rAd expressing a single antigen under the control of an hCMVpromoter that the expression is sufficient to generate significantT-cell and B-cell immune responses. Similarly, expression of twotransgenes expressed by a bidirectional hCMV-rhCMV promoter of thepresent invention is expected to generate a significant T-cell andB-cell immune response to both transgenes. For example, if the twotransgenes encode antigens to elicit an immune response whenadministered to a subject, potent expression of the two transgenes isexpected to generate a measurable immune response against both antigens.

The expression is also very balanced from both sides of thebidirectional hCMV-rhCMV promoter. As used herein, “balancedexpression”, “balance of expression”, “expression balance”, or“balanced” as it refers to expression, mean that the expression from oneside of the bidirectional promoter, as measured for example by differentprotein expression detection techniques such as Western Blot, FACSanalysis, or other assays using luminescence or fluorescence, iscomparable to the expression from the other side of the bidirectionalpromoter. For example, the expression level from one side of thebidirectional hCMV-rhCMV promoter of the present invention is preferablyat least 50%, 60%, 70%, 80%, 90%, or 95% of the expression level fromthe other side of the bidirectional promoter. In certain embodiments,the expression level from one side of the bidirectional hCMV-rhCMVpromoter is about 70-130%, e.g. 80-120%, e.g. 90-110%, e.g. about 100%,of the expression level from the other side of the bidirectionalpromoter. In another example, the ratio of the expression from the twosides of the bidirectional hCMV-rhCMV promoter is in the range of 1/1,1.1/1, 1.2/1, 1.3/1, 1.4/1, 1.5/1, 1.6/1, 1.7/1, 1.8/1, 1.9/1, and 2/1.Furthermore, it is known from rAd expressing a single antigen under thecontrol of an hCMV promoter that the expression is sufficient togenerate significant T-cell and B-cell immune responses. Therefore,balanced expression of two antigens expressed by a bidirectionalhCMV-rhCMV promoter of the present invention could possibly generatecomparable T-cell and B-cell immune response to both antigens, althoughthis may also depend on the expression of the antigen over time and theinherent capability of the antigens themselves to generate certain typesof immune responses. Thus, the bidirectional hCMV-rhCMV promoter of thepresent invention is improved in balance of expression compared to themCMV bidirectional promoter. To compare, there was approximately a10-times higher expression of an antigen positioned at the right side(3′ end) of the bidirectional mCMV promoter compared to the antigenpositioned at the left side (5′ end) of the bidirectional mCMV promoter(which was described in WO 2016/166088), which was already consideredrelatively well-balanced, but clearly is much less balanced than thebidirectional promoter of the instant invention.

An important aspect of vectors, be it DNA vectors such as plasmidvectors or viral vectors such as adenoviral vectors, is the capacity ofthese vectors to accommodate desired transgene sequences. Such capacitymay be limited by size constraints of the vectors, which may forinstance become unstable or even impossible to produce if certain sizelimits are exceeded. The space taken up by a promoter, especially abidirectional promoter that can control expression of more than onetransgene, is therefore an important consideration when designing newvectors, apart from the functional capabilities such promoters shouldhave. The instant bidirectional promoter has the advantage that it isrelatively short, meaning that at a certain size limit of a vector, morespace remains for the transgene, e.g. allowing more epitopes to beincluded if a transgene is an immunogen or allowing expression of largerproteins, as compared to other bidirectional promoters of larger size.

The terms “coding sequence”, “sequence encoding”, or “encoding” are usedinterchangeably herein, and refer to the nucleic acid sequence which istranscribed (DNA) and translated (mRNA) into a polypeptide in vitro orin vivo when operably linked to appropriate regulatory sequences.

A polyadenylation signal, for example the bovine growth hormone polyAsignal (U.S. Pat. No. 5,122,458), may be present behind the transgenes.Preferably, each transgene has a polyA signal, and preferably the polyAsignal for the first transgene is different from the polyA signal forthe second transgene. In one embodiment, a first polyA signal is an SV40polyA signal, and a second polyA signal is the bovine growth hormonepolyA signal.

A sequence comprising an intron may also be placed at one or both endsof the bidirectional promoter of the invention. For example, it is knownthat introns can increase protein expression, in particular in vivo. Anintron as used herein has the normal function and structure as known inthe art, and is a polynucleotide sequence in a nucleic acid that doesnot encode information for protein synthesis and is removed beforetranslation of messenger RNA, by a process known as splicing. An introncomprises a splice donor site (5′end of the intron, usually a GUsequence) and a splice acceptor site (3′end of the intron, usually a GAsequence). A variety of different introns can potentially be usedaccording to the present invention, although it is preferred to userelatively short introns and introns modified to be shorter introns inorder to not take up too much space in a viral vector so that more spaceremains for the transgenes in the recombinant adenovirus. It ispreferred to use a first intron on one side of the bidirectionalpromoter and a second, different intron on the other side of thebidirectional promoter, i.e. each transgene is preceded by a differentintron sequence. In certain embodiments, an intron could be a chimericintron. The skilled person is aware that many different introns areavailable and can be used. However, an advantage of the instant promoteris that it does not require such introns for proper expression, andhence in preferred embodiments there are no introns between the promoterbuilding blocks of the bidirectional promoter and the respectivetransgenes on either side.

The bidirectional promoter of the invention can in certain embodimentsfor instance be used to drive expression of two antigens, with the aimof generating an immune response to these antigens in a vaccineapplication. However, it will be immediately clear to the skilled personthat balanced transgene expression levels can also be relevant fortransgenes for which an immune response is not the primary goal, e.g.for two different transgenes that are used for gene therapy purposes,for expression of heterologous protein complexes, or for proportionalexpression of two antibody chains. Hence, the invention can be practicedwith any combination of transgenes for which expression from a singlerecombinant vector, e.g. adenoviral vector, is desired. Therefore, theidentity of the transgene is not material for the instant invention,which is suitable for example with vectors or adenoviruses comprisingany transgene. Suitable transgenes are well known to the skilled person,and for instance may include transgene open reading frames, for instanceopen reading frames coding for polypeptides that have a therapeuticeffect, e.g. for gene therapy purposes, or polypeptides against which animmune response is desired when the rAd vector is used for vaccinationpurposes. Particularly preferred heterologous nucleic acids are genes ofinterest encoding antigenic determinants towards which an immuneresponse needs to be raised. Such antigenic determinants are alsotypically referred to as antigens. When the recombinant adenovirus isadministered to a subject, an immune response will be raised against theantigen(s). Any desired antigen can be encoded by the adenovirus vector.In typical embodiments according to the invention, antigens arepeptides, polypeptides or proteins from organisms that may cause adisease or condition. Therefore, in a further preferred embodiment, saidheterologous nucleic acid of interest encodes an immunogenic (orantigenic) determinant. More preferably, said immunogenic determinant isan antigen from a bacterium, a virus, yeast or a parasite. The diseasescaused by such organisms are generally referred to as ‘infectiousdisease’ (and are thus not limited to organisms that ‘infect’ but alsoinclude those that enter the host and cause a disease). So-called‘self-antigens’, e.g. tumour antigens, also form part of the state ofthe art, and may be encoded by heterologous nucleic acids in therecombinant adenoviruses according to the present invention.Non-limiting examples from which the antigenic determinants (orantigens) are taken are malaria-causing organisms, such as Plasmodiumfalciparum, tuberculosis-causing organism such as Mycobacteriumtuberculosis, yeasts, or viruses. In other preferred embodiments,antigens from viruses such as flaviviruses (e.g., West Nile Virus,Hepatitis C Virus, Japanese Encephalitis Virus, Dengue Virus), Ebolavirus, Human Immunodeficiency Virus (HIV), and Marburg virus may be usedin compositions according to the present invention. In one embodiment,said antigen is the CS protein or immunogenic part thereof from P.falciparum (for examples of adenovirus vectors encoding CS, see e.g.(Havenga et al., 2006; Ophorst et al., 2006); WO 2004/055187, allincorporated in their entirety by reference herein). In anotherembodiment, the antigenic determinant is a protein of one antigen-, or afusion protein of several antigens from M. tuberculosis, such as theAg85A, Ag85B and/or the TB10.4 proteins or immunogenic part(s) thereof(see for the construction and production of such TB vaccine viruses e.g.WO 2006/053871, incorporated by reference herein). In yet anotherembodiment, said antigenic determinant is a viral glycoprotein orimmunogenic part thereof, such as GP from a filovirus, such as Ebolavirus or Marburg virus (e.g. (Geisbert et al., 2011; Sullivan et al.,2006; Sullivan et al., 2003). In yet further embodiments, saidimmunogenic determinant is from an HIV protein such as gag, pol, env,nef, or variants thereof (for examples of adenovirus based HIV vaccines,see e.g. WO 2009/026183, WO 2010/096561, WO 2006/120034, WO 02/22080, WO01/02607). In other embodiments, said antigenic determinant is a HA, NA,M, or NP protein, or immunogenic part of any of these, from influenzavirus (e.g. (Hu et al., 2011; Zhou et al., 2010); review by (Vemula &Mittal, 2010)). In other embodiments, the antigenic determinant is a HAprotein or immunogenic part thereof from a measles virus (e.g. WO2004/037294). In other embodiments, the antigenic determinant is rabiesvirus glycoprotein (e.g. (Zhou, Cun, Li, Xiang, & Ertl, 2006)). Infurther embodiments, the antigen is from a respiratory syncytial virus(RSV), e.g. RSV F protein (see e.g. WO 2013/139911 and WO 2013/139916),or RSV G protein, or both, or other RSV proteins. In other embodiments,the antigen is from another virus such as human papillomavirus or otherviruses, etc. The recombinant adenovirus may encode two differentantigens from the same organism. The recombinant adenovirus may alsoencode combinations of antigens from different organisms, e.g. a firstantigen from a first organism and second antigen from a second organism.It is also possible to encode an antigen and for instance an adjuvantinto the same adenovirus, e.g. an antigen and a Toll-Like-Receptor (TLR)agonist, such as a TLR3 agonist, such as dsRNA or a mimetic thereof orthe like (e.g. WO 2007/100908). In certain embodiments, the recombinantvector, e.g. recombinant adenovirus, encodes two different antigens,each under control of the bidirectional hCMV-rhCMV promoter. In otherembodiments, the vector or recombinant (adeno)virus encodes an antigenand an immune modulator, each under control of the bidirectionalhCMV-rhCMV promoter. In certain embodiments, further heterologoussequences or transgenes may be present in the vector or recombinant(adeno)virus, besides the first and second transgene that are undercontrol of the bidirectional hCMV-rhCMV promoter.

The invention also provides a method for producing a genetically stablerecombinant adenovirus comprising a first and a second transgene thateach are potently expressed when the adenovirus infects a target cell,the method comprising: preparing a construct comprising a bidirectionalhCMV-rhCMV promoter operably linked to a first transgene in onedirection and to a second transgene in the opposite direction, andincorporating said construct into the genome of the recombinantadenovirus. The preparation of the construct as such encompasses the useof standard molecular cloning methods that are well known (see e.g.(Holterman et al., 2004; Lemckert et al., 2006; Vogels et al., 2003);Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual,2nd edition, 1989; Current Protocols in Molecular Biology, Ausubel F M,et al, eds, 1987; the series Methods in Enzymology (Academic Press,Inc.); PCR2: A Practical Approach, MacPherson MJ, Hams B D, Taylor G R,eds, 1995), as known to the skilled person and routinely performed inthe field of recombinant adenovirus technology, and exemplified herein.The bidirectional hCMV-rhCMV promoter has the features as describedabove, and can be obtained by routine methods. For convenience, theskilled person may manipulate the adenovirus genome by cloning intosmaller fragments, e.g. a first part for the left part of the genome upto the E1 region for easy manipulation and introduction of thetransgenes in plasmid form and a second, larger, part for the remainderof the genome that can upon recombination with the first part result ina complete adenovirus genome (see e.g. WO 99/55132).

The rAd of the present invention has the advantage that it can expresstwo transgenes and remains genetically stable, unlike adenovirusesprepared by the various alternative approaches for expressing twotransgenes that are provided in the prior art, while also providingbalanced expression of the two transgenes driven by the bidirectionalpromoter. Thus, the bidirectional hCMV-rhCMV promoter solves the problemof genetic instability of adenoviruses that express two transgenes, andof imbalanced expression of the two transgenes, and due to itsrelatively small size allows significantly more space for the transgenesequences than certain other bidirectional promoters that have a largersize (e.g. it is about 1 kb shorter than the mCMV bidirectionalpromoter, thus in theory a given vector with a size capacity constraintcould accommodate transgenes that in total are about 1 kb longer thanthe same vector wherein the transgenes would be driven by the mCMVpromoter).

To test genetic stability, rAd are rescued and propagated in anappropriate cell line, e.g., helper cell line PER.C6®. Viral DNA isisolated at certain passage numbers and the integrity of the rAd genomecan be analyzed by one or more of the following: PCR analysis forpresence of the transgene region or absence of deletion bands,restriction digests of the rAd genome for presence or absence ofdifferences in restriction fragments, and/or sequencing of the rAdgenome or of PCR-products of the rAd genome for presence or absence ofmutations in the rAd sequences. With regard to the rAd of the presentinvention, “genetically stable” means that the nucleotide sequence doesnot change from the plasmids used for generation of the rAd to laterproduction stages of the rAd, such that rAd expressing two transgeneshas the same genetic stability as a comparable rAd with a singletransgene (e.g., behind a hCMV promoter) as suitable for large scalebatch productions. For example, PCR analysis using primers flanking theexpression cassette does not show deletion fragments (bands) compared toearlier passage numbers of the rAd or the starting material and/orsequencing the PCR product of the E1, E3 and E4 regions confirms thatthe nucleotide sequence does not change. Preferably sequencing theregion containing the expression cassette with the bidirectionalpromoter confirms that the nucleotide sequence does not change in theregion containing the expression cassette.

Genetic stability is thoroughly assessed in this study compared to othertesting methods such as test digestions on a single produced virusbatch. Sensitivity of the assay is increased by the following means:several viral populations (plaques) are isolated and subjected toextended passaging. The extended passaging, combined with a PCR analysisusing primers flanking the expression cassette allows for detection of asmall proportion of deletion mutants in the rAd population which mightbe overlooked using other methods. Further, sequencing analysis isperformed to exclude occurrence of point mutations, such as introductionof stop codons in the open reading frame of the transgene. Morespecifically, since viral mutations always present a chance event, oneplaque may be stable whereas another one may present a deletion band.Therefore, to correctly assess genetic stability, several viralpopulations (plaques) need to be tested. In case a mutation occurs,which enables the vector to replicate more efficiently than the parentalvector, this can lead to outgrowth of the mutant version, which is oftenonly observed following extended passaging as described in this study.Preferably, the rAd of the present invention are genetically stable forat least up to 10 passages, and even more preferably for at least up to13 passages in the test system used, such that the virus is sufficientlystable for large scale production campaigns. It was recently found thata recombinant adenovirus that has two transgenes that are under controlof the bidirectional mCMV promoter is genetically stable, see e.g., WO2016/166088 (which also describes that various other solutions that hadbeen described in the art for expression of two antigens from onevector, failed to lead to stable rAd or potent expression, so that themCMV promoter was described therein as the most preferred solution forthis problem). The instant application demonstrates that a recombinantadenovirus that has two transgenes that are under control of thebidirectional hCMV-rhCMV promoter of the invention are also geneticallystable, and moreover have a more balanced expression of the twotransgenes compared to the situation where they are under control of thebidirectional mCMV promoter.

The recombinant adenovirus produced according to the methods of theinvention can be prepared according to the embodiments described abovefor the recombinant adenovirus.

The invention also provides a method for expressing at least twotransgenes in a cell, the method comprising providing the cell with avector or a recombinant virus, e.g. a recombinant adenovirus, accordingto the invention. Providing a cell with a recombinant adenovirus can bedone via administration of the adenovirus to a subject, or viaintroduction (e.g. infection) of the adenovirus in vitro or ex vivo intoa cell. In certain embodiments the invention provides a recombinantadenoviral vector for use in expressing at least two transgenes in acell, e.g. by administering the recombinant adenovirus to a subject.

The invention also provides a method for inducing an immune responseagainst at least two antigens, comprising administering to a subject avector, e.g. a recombinant (adeno)virus according to the invention. Theinvention also provides a vector or a recombinant (adeno)virus accordingto the invention for use in inducing an immune response against at leasttwo antigens.

The invention also provides a recombinant DNA molecule comprising thebidirectional hCMV-rhCMV promoter of the present invention or the genomeof a recombinant adenovirus of the invention. The skilled person will beaware that this may also be a combination of at least two differentrecombinant DNA molecules that together can form the single recombinantDNA molecule of the invention. Such molecules are useful in manipulatingthe genome and creating novel recombinant adenoviruses. The genomeencodes the proteins that are required for adenovirus replication andpackaging in permissive cells.

The term ‘about’ for numerical values as used in the present disclosuremeans the value ±10%.

Producer cells are cultured to increase cell and virus numbers and/orvirus titers. Culturing a cell is done to enable it to metabolize,and/or grow and/or divide and/or produce virus of interest according tothe invention. This can be accomplished by methods such as well-known topersons skilled in the art, and includes but is not limited to providingnutrients for the cell, for instance in the appropriate culture media.Suitable culture media are well known to the skilled person and cangenerally be obtained from commercial sources in large quantities, orcustom-made according to standard protocols. Culturing can be done forinstance in dishes, roller bottles or in bioreactors, using batch,fed-batch, continuous systems and the like. Suitable conditions forculturing cells are known (see e.g. Tissue Culture, Academic Press,Kruse and Paterson, editors (1973), and R. I. Freshney, Culture ofanimal cells: A manual of basic technique, fourth edition (Wiley-LissInc., 2000, ISBN 0-471-34889-9).

Typically, the adenovirus will be exposed to the appropriate producercell in a culture, permitting uptake of the virus. Usually, the optimalagitation is between about 50 and 300 rpm, typically about 100-200, e.g.about 150, typical DO is 20-60%, e.g. 40%, the optimal pH is between 6.7and 7.7, the optimal temperature between 30 and 39° C., e.g. 34-37° C.,and the optimal MOI between 5 and 1000, e.g. about 50-300. Typically,adenovirus infects producer cells spontaneously, and bringing theproducer cells into contact with rAd particles is sufficient forinfection of the cells. Generally, an adenovirus seed stock is added tothe culture to initiate infection, and subsequently the adenoviruspropagates in the producer cells. This is all routine for the personskilled in the art.

After infection of an adenovirus, the virus replicates inside the celland is thereby amplified, a process referred to herein as propagation ofadenovirus. Adenovirus infection results finally in the lysis of thecells being infected. The lytic characteristics of adenovirus thereforepermits two different modes of virus production. The first mode isharvesting virus prior to cell lysis, employing external factors to lysethe cells. The second mode is harvesting virus supernatant after(almost) complete cell lysis by the produced virus (see e.g. U.S. Pat.No. 6,485,958, describing the harvesting of adenovirus without lysis ofthe host cells by an external factor). It is preferred to employexternal factors to actively lyse the cells for harvesting theadenovirus.

Methods that can be used for active cell lysis are known to the personskilled in the art, and have for instance been discussed in WO 98/22588,p. 28-35. Useful methods in this respect are for example, freeze-thaw,solid shear, hypertonic and/or hypotonic lysis, liquid shear,sonication, high pressure extrusion, detergent lysis, combinations ofthe above, and the like. In one embodiment of the invention, the cellsare lysed using at least one detergent. Use of a detergent for lysis hasthe advantage that it is an easy method, and that it is easily scalable.

Detergents that can be used, and the way they are employed, aregenerally known to the person skilled in the art. Several examples arefor instance discussed in WO 98/22588, p. 29-33. Detergents can includeanionic, cationic, zwitterionic, and nonionic detergents. Theconcentration of the detergent may be varied, for instance within therange of about 0.1%-5% (w/w). In one embodiment, the detergent used isTriton X-100.

Nuclease may be employed to remove contaminating, i.e. mostly from theproducer cell, nucleic acids. Exemplary nucleases suitable for use inthe present invention include Benzonase®, Pulmozyme®, or any other DNaseand/or RNase commonly used within the art. In preferred embodiments, thenuclease is Benzonase®, which rapidly hydrolyzes nucleic acids byhydrolyzing internal phosphodiester bonds between specific nucleotides,thereby reducing the viscosity of the cell lysate. Benzonase® can becommercially obtained from Merck KGaA (code W214950). The concentrationin which the nuclease is employed is preferably within the range of1-100 units/ml. Alternatively, or in addition to nuclease treatment, itis also possible to selectively precipitate host cell DNA away fromadenovirus preparations during adenovirus purification, using selectiveprecipitating agents such as domiphen bromide (see e.g. U.S. Pat. No.7,326,555; (Goerke, To, Lee, Sagar, & Konz, 2005); WO 2011/045378; WO2011/045381).

Methods for harvesting adenovirus from cultures of producer cells havebeen extensively described in WO 2005/080556.

In certain embodiments, the harvested adenovirus is further purified.Purification of the adenovirus can be performed in several stepscomprising clarification, ultrafiltration, diafiltration or separationwith chromatography as described in for instance WO 05/080556,incorporated by reference herein. Clarification may be done by afiltration step, removing cell debris and other impurities from the celllysate. Ultrafiltration is used to concentrate the virus solution.Diafiltration, or buffer exchange, using ultrafilters is a way forremoval and exchange of salts, sugars and the like. The person skilledin the art knows how to find the optimal conditions for eachpurification step. Also, WO 98/22588, incorporated in its entirety byreference herein, describes methods for the production and purificationof adenoviral vectors. The methods comprise growing host cells,infecting the host cells with adenovirus, harvesting and lysing the hostcells, concentrating the crude lysate, exchanging the buffer of thecrude lysate, treating the lysate with nuclease, and further purifyingthe virus using chromatography.

Preferably, purification employs at least one chromatography step, asfor instance discussed in WO 98/22588, p. 61-70. Many processes havebeen described for the further purification of adenoviruses, whereinchromatography steps are included in the process. The person skilled inthe art will be aware of these processes, and can vary the exact way ofemploying chromatographic steps to optimize the process. It is forinstance possible to purify adenoviruses by anion exchangechromatography steps, see for instance WO 2005/080556. Many otheradenovirus purification methods have been described and are within thereach of the skilled person. Further methods for producing and purifyingadenoviruses are disclosed in for example WO 00/32754, WO 04/020971, WO2006/108707, and U.S. Pat. Nos. 5,837,520 and 6,261,823, allincorporated by reference herein.

For administering to humans, the invention may employ pharmaceuticalcompositions comprising the vector or recombinant virus, e.g., rAd, anda pharmaceutically acceptable carrier or excipient. In the presentcontext, the term “Pharmaceutically acceptable” means that the carrieror excipient, at the dosages and concentrations employed, will not causeany unwanted or harmful effects in the subjects to which they areadministered. Such pharmaceutically acceptable carriers and excipientsare well known in the art (see Remington's Pharmaceutical Sciences, 18thedition, A. R. Gennaro, Ed., Mack Publishing Company [1990];Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook ofPharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]). The purified rAd preferably is formulated andadministered as a sterile solution although it is also possible toutilize lyophilized preparations. Sterile solutions are prepared bysterile filtration or by other methods known per se in the art. Thesolutions are then lyophilized or filled into pharmaceutical dosagecontainers. The pH of the solution generally is in the range of pH 3.0to 9.5, e.g. pH 5.0 to 7.5. The rAd typically is in a solution having asuitable buffer, and the solution of rAd may also contain a salt.Optionally stabilizing agent may be present, such as albumin. In certainembodiments, detergent is added. In certain embodiments, rAd may beformulated into an injectable preparation. These formulations containeffective amounts of rAd, are either sterile liquid solutions, liquidsuspensions or lyophilized versions and optionally contain stabilizersor excipients. An adenovirus vaccine can also be aerosolized forintranasal administration (see e.g. WO 2009/117134).

For instance adenovirus may be stored in the buffer that is also usedfor the Adenovirus World Standard (Hoganson et al., 2002): 20 mM Tris pH8, 25 mM NaCl, 2.5% glycerol. Another useful formulation buffer suitablefor administration to humans is 20 mM Tris, 2 mM MgCl₂, 25 mM NaCl,sucrose 10% w/v, polysorbate-80 0.02% w/v. Another formulation bufferthat is suitable for recombinant adenovirus comprises 10-25 mM citratebuffer pH 5.9-6.2, 4-6% (w/w) hydroxypropyl-beta-cyclodextrin (HBCD),70-100 mM NaCl, 0.018-0.035% (w/w) polysorbate-80, and optionally0.3-0.45% (w/w) ethanol. Obviously, many other buffers can be used, andseveral examples of suitable formulations for the storage and forpharmaceutical administration of purified (adeno)virus preparations areknown, including those that can for instance be found in EP0853660, U.S.Pat. No. 6,225,289 and in WO 99/41416, WO 99/12568, WO 00/29024, WO01/66137, WO 03/049763, WO 03/078592, WO 03/061708.

In certain embodiments a composition comprising the adenovirus furthercomprises one or more adjuvants. Adjuvants are known in the art tofurther increase the immune response to an applied antigenicdeterminant, and pharmaceutical compositions comprising adenovirus andsuitable adjuvants are for instance disclosed in WO 2007/110409,incorporated by reference herein. The terms “adjuvant” and “immunestimulant” are used interchangeably herein, and are defined as one ormore substances that cause stimulation of the immune system. In thiscontext, an adjuvant is used to enhance an immune response to theadenovirus vectors of the invention. Examples of suitable adjuvantsinclude aluminium salts such as aluminium hydroxide and/or aluminiumphosphate; oil-emulsion compositions (or oil-in-water compositions),including squalene-water emulsions, such as MF59 (see e.g. WO 90/14837);saponin formulations, such as for example QS21 and ImmunostimulatingComplexes (ISCOMS) (see e.g. U.S. Pat. No. 5,057,540; and WO 90/03184,WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbialderivatives, examples of which are monophosphoryl lipid A (MPL),3-O-deacylated MPL (3dMPL), CpG-motif containing oligonucleotides,ADP-ribosylating bacterial toxins or mutants thereof, such as E. coliheat labile enterotoxin LT, cholera toxin CT, and the like. It is alsopossible to use vector-encoded adjuvant, e.g. by using heterologousnucleic acid that encodes a fusion of the oligomerization domain ofC4-binding protein (C4 bp) to the antigen of interest (Ogun,Dumon-Seignovert, Marchand, Holder, & Hill, 2008), or heterologousnucleic acid encoding a toll-like receptor (TLR) agonist, such as a TLR3agonist such as dsRNA (see e.g. WO 2007/100908) or the like. Such rAdaccording to the invention may for instance encode an antigen ofinterest on one side of the bidirectional promoter and a TLR3 agonist onthe other side of the bidirectional promoter. Such rAd are particularlysuited for administration via a mucosal route, e.g. oral administration(see e.g. WO 2007/100908). In certain embodiments the compositions ofthe invention comprise aluminium as an adjuvant, e.g. in the form ofaluminium hydroxide, aluminium phosphate, aluminium potassium phosphate,or combinations thereof, in concentrations of 0.05-5 mg, e.g. from0.075-1.0 mg, of aluminium content per dose.

In other embodiments, the compositions do not comprise adjuvants.

A pharmaceutical composition according to the invention may in certainembodiments be a vaccine.

Adenovirus compositions may be administered to a subject, e.g. a humansubject. The total dose of the adenovirus provided to a subject duringone administration can be varied as is known to the skilledpractitioner, and is generally between 1×10⁷ viral particles (vp) and1×10¹² vp, preferably between 1×10⁸ vp and 1×10¹¹ vp, for instancebetween 3×10⁸ and 5×10¹⁰ vp, for instance between 10⁹ and 3×10¹⁰ vp.

Administration of adenovirus compositions can be performed usingstandard routes of administration. Non-limiting embodiments includeparenteral administration, such as by injection, e.g. intradermal,intramuscular, etc, or subcutaneous or transcutaneous, or mucosaladministration, e.g. intranasal, oral, and the like. In one embodiment acomposition is administered by intramuscular injection, e.g. into thedeltoid muscle of the arm, or vastus lateralis muscle of the thigh. Theskilled person knows the various possibilities to administer acomposition, e.g. a vaccine in order to induce an immune response to theantigen(s) in the vaccine.

A subject as used herein preferably is a mammal, for instance a rodent,e.g. a mouse, or a non-human-primate, or a human. Preferably, thesubject is a human subject.

It is also possible to provide one or more booster administrations ofone or more adenovirus vaccines. If a boosting vaccination is performed,typically, such a boosting vaccination will be administered to the samesubject at a moment between one week and one year, preferably betweentwo weeks and four months, after administering the composition to thesubject for the first time (which is in such cases referred to as‘priming vaccination’). In alternative boosting regimens, it is alsopossible to administer different vectors, e.g. one or more adenovirusesof different serotype, or other vectors such as MVA, or DNA, or protein,to the subject as a priming or boosting vaccination.

Various publications, which may include patents, published applications,technical articles and scholarly articles, are cited throughout thespecification in parentheses, and full citations of each may be found atthe end of the specification. Each of these cited publications isincorporated by reference herein, in its entirety.

EXAMPLES

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative methods and examples, make and utilize the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out certain embodiments,features, and advantages of the present invention, and are not to beconstrued as limiting in any way the remainder of the disclosure. Theexamples merely serve to clarify the invention.

Methods Cell Culture:

PER.C6® cells (Fallaux et al., 1998) were maintained in Dulbecco'smodified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS),supplemented with 10 mM MgCl₂.

Adenovirus Vector Construction in pAdApt35 and Pshuttle26 Plasmids

Different bidirectional promoter constructs were cloned into pAdApt35(Vogels et al. 2007) or pshuttle26 plasmids. Pshuttle26 was constructedbased on the previously described pAdapt26 plasmid (Abbink et al.,2007). A 2-Kb fragment containing the right part of the Ad26 vectorgenome was synthesized and subcloned into pAdApt26.Luc of which the SpeIsite in the CMV promoter had first been disrupted by introduction of asingle bp substitution. As a result pshuttle26 can be used to constructan adenovirus vector by homologous recombination with an Ad26 cosmid orby homologous recombination with an Ad26 full-length genome plasmid.

Since the pAdapt35 and pshuttle26 plasmids only harbor a standardunidirectional expression cassette with one promoter and one SV40derived polyA signal, restriction sites to place another transgene plusBGH polyA signal were added by fusion PCR. The fusion PCR productcontaining SpeI, NotI—BGH polyA-EcoRI—Luciferase—KpnI, SalI, AvrII wasinserted into the plasmids in the correct orientation by molecularcloning via SpeI and AvrII restriction sites. As a result, theunidirectional hCMV promoter could be replaced by the bidirectionalpromoter sequences using the flanking restriction sites AvrII andHindIII. Transgenes were placed on both sides of the bidirectionalpromoter using the HindIII and XbaI restriction sites on one side andAvrII, SalI or KpnI on the other side (FIG. 3A). The selection of AvrII,SalI or KpnI was dependent on the uniqueness of restriction sites in theplasmid sequence. The complete bidirectional expression cassettes withthe different bidirectional promoter constructs were cloned inpShuttle26 plasmids and transferred to pAdapt35 plasmids using SpeI orNotI and XbaI restriction sites. A Kozak sequence (5′ GCCACC 3′) wasincluded directly in front of each ATG start codon, and two stop codons(5′ TGA TAA 3′) were added at the end of each coding sequence. Asdescribed herein, the recombinant adenoviruses and vectors are referredto generally as rAd or rAd vectors and more specifically as rAd35 orrAd26 and associated vectors.

Adenovirus Generation, Infections and Propagation.

All adenoviruses were generated in PER.C6 cells by homologousrecombination and produced as previously described (for rAd35: (Havengaet al., 2006); for rAd26: (Abbink et al., 2007)). Briefly, PER.C6 cellswere transfected with rAd vector encoding plasmids, using Lipofectamineaccording to the instructions provided by the manufacturer (LifeTechnologies). For rescue of rAd35 vectors, the pAdApt35 plasmids andpWE/Ad35.pIX-rITR.dE3.5orf6 cosmid were used, whereas for rAd26 vectors,the pShuttle26 plasmids and pWE.Ad26.dE3.5orf6 cosmid were used. Cellswere harvested one day after full cytopathic effect (CPE),freeze-thawed, centrifuged for 5 min at 3,000 rpm, and stored at −20° C.Next the viruses were plaque purified and amplified in PER.C6 cellscultured on a single well of a multiwell 24 tissue culture plate.Further amplification was carried out in PER.C6 cells cultured using aT25 tissue culture flask.

Expression Analysis

To evaluate potency of expression and expression balance, viral vectorswere generated with reporter genes encoding enhanced Green FluorescentProtein (eGFP protein accession number AAB02572.1) and FireflyLuciferase (Luciferase protein accession number ACH53166). The relativeeGFP mean fluorescence intensity (MFI) and Luciferase relative lightunits (RLU) were recorded for each promoter and reporter genecombination with HEK293 cells (transient transfection with pAdAptvectors or pshuttle vectors) or A549 cells (virus infection). Luciferaseactivity was measured in cell lysates in presence with 0.1% DTT (1M), inLuminoskan™ Ascent Microplate Luminometer. The eGFP fluorescence wasmeasured in the flow cytometer (FACS) by, trypsinizing, centrifuging,and re-suspending cell pellets in PBS/1% FBS (non-virus material) or inCellFix (virus material).

Genetic Stability Testing of Adenoviral Vectors in PER.C6 Cells.

Genetic stability testing of the vaccine vectors was performed to ensuregenetic stability in the production process, which involves severalpassages in PER.C6 cells. Generation, plaque purification, and expansionto T25 format of the recombinant vaccine vectors was achieved asdescribed above. Briefly, recombinant viruses were generated by plasmidtransfections in the E1-complementing cell line PER.C6 andplaque-purified. 5 plaques were selected for up-scaling from multiwell24 (MW24) to a T25 flask. Subsequently, new PER.C6 cells were infectedin T25 format until viral passage number 13. The propagation of theviruses was performed using a predetermined infectious volume that wouldgive full cytopathic effect 2 days post infection, which wasretrospectively determined to be in a range of virus particle per cellratio of 50 for rAd35 and 900 for rAd26. Viral DNA was isolated from p13material and tested for presence of the complete transgene expressioncassette by PCR analysis. The vaccine vectors were propagated up topassage number 13 in PER.C6 cells. The propagation was performed in away to give full CPE two days post infection. rAd35 viruses wereharvested 2 days after full CPE, whereas rAd26 viruses were harvestedone day after full CPE. Viral DNA was isolated at passage 2, passage 5,passage 10 and passage 13 and absence of deletions was tested by PCRanalysis using primers that flank the transgene expression cassette.Absence of deletion mutants was defined by the following parameters:Band size of PCR product corresponds to positive control (PCR product ofplasmid used for virus rescue), no additional bands below the expectedPCR product (unless additional bands show to be unspecific PCR productsbecause they are also present in the positive control), approved assay:no band in the PCR H₂O control. To further confirm genetic stability thePCR product of the expression cassette plus flanking regions of someplaques were sequenced.

Example 1: Bidirectional Promoter Construct Design

The potent bidirectional mouse CMV (mCMV) promoter was identified as auseful promoter for expression of two antigens from a bidirectionalexpression cassette in E1 region of adenoviral vectors in previous work,WO 2016/166088. While the vectors harbouring the mCMV bidirectionalpromoter expressed the antigens, were genetically stable and induced animmune response against both encoded antigens, antigen expression andthe induced immune response were not balanced as explained in thefollowing. Expression of the antigen placed on the right side of thebidirectional promoter was higher than of the antigen placed on the leftside of the bidirectional promoter, resulting in a higher immuneresponse against the antigen placed on the right side of thebidirectional promoter. The difference in expression levels for mCMVbidirectional was ca. 10 fold. However, in order to substitute the mixof two vectors expressing only one antigen, for certain applications abalanced bidirectional promoter is desirable which induces comparablelevels of antigen expression of both antigens. In addition, it would bebeneficial if the size of a bidirectional promoter would be relativelysmall.

In order to identify a potent and more balanced bidirectional promoter apanel of new bidirectional promoters was designed. Bidirectionalpromoter designs with small size with a maximum size of 2 kB werepreferred in order to retain sufficient space for antigens due to theoverall size restriction of adenoviral vectors. Further, building blockswithout extensive stretches of sequence identities (<15 nucleotides)were preferred in order to prevent deletions by homologous recombinationin the adenoviral vector. Bidirectional promoters of the presentinvention direct gene expression in a bidirectional fashion controllingexpression of the genes placed on both sides of the bidirectionalpromoter sequence. These bidirectional promotors are continuous generegulatory sequences that contain enhancer elements and intron elementsbesides the promoter elements and are defined by the building blocks asdescribed herein. The building blocks used for design of the syntheticbidirectional promoters are derived from known potent unidirectionalpromoters, enhancers and intron sequences. Promoters drive expression ofone gene placed downstream of the promoter sequence and typicallycontain a TATA box sequence and the transcription start site (TSS).Enhancer sequences can enhance gene expression from a promoter. Intronsequences have been described to increase gene expression in vitro andespecially in vivo.

The following panel of bidirectional promoter constructs were designedand tested:

1. rCMV-hEF1α I (FIG. 1A, SEQ ID NO: 1) 2. rCMV-hEF1α II (FIG. 1B, SEQID NO: 2) 3. rhCMV-CAG1 (FIG. 1C, SEQ ID NO: 3) 4. hCMV-rhCMV (FIG. 1D,SEQ ID NO: 4) 5. rCMV-CAG (FIG. 1E, SEQ ID NO: 5) 6. rhCMV-CAG2 (FIG.1F, SEQ ID NO: 6) 7. rCMV bidir 1 (FIG. 1G, SEQ ID NO: 10) 8. rCMV bidir2 (FIG. 1H, SEQ ID NO: 8) 9. rCMV bidir 1.1 (FIG. 1I, SEQ ID NO: 7) 10.hCMV-CAG4 (FIG. 1J, SEQ ID NO: 11)Other bidirectional promoter constructs were also tested in a previouspatent application (PCT/EP2016/057982):

1. mCMV bidir.

2. hCMV-CAG

3. mCMV-CAG

Schematic representations of the bidirectional promoter designs andtheir building blocks are shown in FIG. 1 and described in more detailbelow:

The promoter and enhancer building blocks used in the different designsare derived from cytomegalovirus immediate early (IE) regions (typicallyreferred to herein as the hCMV promoter and the hCMV enhancer andsometimes also referred to as hCMV IE), the chicken beta actin/rabbitbeta globin promoter sequence and the human elongation factor 1 αpromoter (hEF1α promoter) sequence. The introns are derived fromchimeric chicken beta actin/rabbit beta globin sequence, hEF1α firstintron and the human apolipoprotein A-1 intron (hApoA1 intron).

The human cytomegalovirus major immediate early promoter is known as apotent promoter in various mammalian cell lines (Powell et al., 2015).hCMV and other herpesviruses express genes in three phases, immediateearly (IE), early and late phase. The major immediate early promotersactivate heterologous genes at high levels in various mammalian celllines.

While the human cytomegalovirus major immediate early promoter andenhancer are most frequently used in design of transgene expressioncassettes (Addison, Hitt, Kunsken, & Graham, 1997; C. Harro et al.,2009), major immediate early promoters and enhancers ofcytomegaloviruses infecting other species such as mouse (mCMV) (Addisonet al., 1997; Chatellard et al., 2007; C. Harro et al., 2009), rat(rCMV) (Sandford & Burns, 1996; Voigt, Sandford, Hayward, & Burns, 2005)and rhesus monkeys (rhCMV) (Barry, Alcendor, Power, Kerr, & Luciw, 1996;Chan, Chiou, Huang, & Hayward, 1996; Chang et al., 1993; Hansen,Strelow, Franchi, Anders, & Wong, 2003) are also known and can be usedin the design of potent expression cassettes. Specifically, the rhesusCMV sequence is derived from the major immediate early region of theCercopithecine herpesvirus 5. The rhesus CMV short promoter portion wasidentified by alignments with hCMV and chimpanzee CMV short promoters.The hEF1α promoter is also described to be a potent promoter sequencefor heterologous gene expression in mammalian cells (Kim, Uetsuki,Kaziro, Yamaguchi, & Sugano, 1990).

The chimeric promoter CAG consisting of the hCMV enhancer, the chickenbeta actin promoter (A) and a hybrid chicken beta actin/rabbit betaglobin intron (G) sequence is described to be a potent promoter forexpression of heterologous genes, can be shortened and can be utilizedfor expression of antigens (Richardson et al., 2009). The study byRichardson et al. describes a modification of the CAG promoter resultingin the Δ829CAG promoter version in which the hybrid intron issignificantly shortened. This Δ829CAG promoter version without the hCMVenhancer (Δ829AG) was used as a building block for design of certainbidirectional promoters, denoted as the AG portion in the drawings, butreferred to as CAG in the names of the bidirectional promoters andreferred to as CAG or AG throughout the text.

In the following the arrangement of the building blocks to design thebidirectional promoter sequences is described:

The shortened AG promoter and hEF1α promoter harbor introns as crucialparts of the described potent regulatory sequences. Where an AG promoteror an hEF1α promoter was used in the bidirectional promoter design, wealso placed a heterologous intron sequence on the opposite side of thebidirectional promoter design. Different potential bidirectionalpromoter designs of the rCMV immediate early promoter were made based onthe natural bidirectionality of the mouse CMV promoter.

It has been described previously that a synthetic combination of anhEF1α derived intron and an mCMV promoter sequence yields a potentregulatory sequence. Therefore, hEF1α sequences were combined withelements of the rCMV promoter (another promoter derived from amuromegalovirus like the mCMV) and enhancer to design the bidirectionalpromoter sequences rCMV-hEF1α I and rCMV-hEF1α II (see, for example,FIGS. 1A and 1B). Since the hEF1α intron is described to increaseexpression levels but is a very long sequence, our designs haveattempted to significantly shorten the hEF1α intron sequence as isdescribed by experimental approach for the CAG promoter (Richardson etal., 2009). To this end, part of the intron sequence was removed whilepreserving the described splice donor, splice acceptor and putativebranch point site plus described cellular factor binding sites. Twodifferent versions of the shortened intron were designed in which thepromoter/intron combination hEF1α I preserves more sites than thepromoter/intron combination hEF1α II, yielding the bidirectionalpromoters rCMV-hEF1α I and rCMV-hEF1α II.

Additional bidirectional promoter designs are based on the shortened AGpromoter in combination with enhancer and promoter sequences ofcytomegalovirus major immediate early promoters derived from differentspecies, including rhesus CMV (rhCMV), rat CMV (rCMV) and human CMV(hCMV). These bidirectional promoters are called rhCMV-CAG1, rhCMV-CAG2,hCMV-CAG4 and rCMV-CAG. The difference between rhCMV-CAG1 and rhCMV-CAG2is the orientation of the rhCMV enhancer sequence.

An additional bidirectional promoter design was based on a combinationof an hCMV enhancer, a human CMV promoter (hCMV), and a rhesus CMVpromoter (rhCMV). The resulting bidirectional promoter construct isreferred to as hCMV-rhCMV throughout the text.

Since it was previously described (Amendola et al, 2005) that thearrangement of one enhancer flanked by two promoters results incoordinate expression of two genes of interest, the use of strongpromoter and enhancer building blocks should theoretically result inbidirectional promoters of comparable potency and balance.

Besides the synthetic bidirectional promoter designs, a potentiallynatural bidirectional promoter derived from the rCMV mIE region was usedas a basis for the bidirectional promoter sequences rCMV bidir 1 andrCMV bidir 2 and rCMV bidir 1.1. While all three bidirectional promoterdesigns harbor a putative minimal rCMV promoter and a putative minimalrCMV vOX2 promoter flanking an rCMV enhancer sequence, the designsdiffer in the length of the enhancer fragment and the orientation of theenhancer fragment. The vOX2 promoter is driving transcription of the ratcellular CD200 (vOX2) gene immediately to the right of the MIE region(Voigt et al., 2005).

Example 2: Screening of Different Promoter Constructs for Potent andBalanced Expression of Reporter Genes

In a first screening experiment, expression from different bidirectionalpromoter constructs was evaluated with transient transfections in HEK293using reporter genes Luciferase and eGFP for a quantitative potencyreadout. For the transient transfections of pAdapt35 plasmids, thebidirectional promoters had the Luciferase transgene on the left sideand the eGFP transgene on the right side of the bidirectional promoter.

Three independent transfection experiments were performed with plasmidsharbouring the different bidirectional promoter designs. In experiment 1(FIG. 2A) the promoters rCMV bidir.2, rCMV-hEF1α II, rhCMV-hEF1α I,rhCMV-CAG1 and hCMV-rhCMV were tested. While rCMV bidir.2, rCMV-EF1 α IIand rhCMV-CAG1 invariably display bidirectional promoter function albeitless potent than the hCMV promoter with unidirectional control (SEQ IDNO:9), rCMV-hEF1α I unexpectedly does not display promoter potency abovethe negative control for eGFP expression and very poor promoter potencyfor luciferase expression. In experiment 1, hCMV-rhCMV is the mostpotent bidirectional promoter. Surprisingly, hCMV-rhCMV is a potentbidirectional promoter with very balanced transgene expression, albeitwith slightly lower expression levels than the unidirectional hCMVpromoter. In the screening experiment 2 (FIG. 2B) the promotershCMV-CAG4, rCMV bidir.1.1, rCMV-CAG were tested. The hCMV-CAG4 promoterdisplayed potent promoter activity on both sides, which was comparableto or even higher than the unidirectional hCMV control in thisexperiment. The rCMV bidir.1.1 promoter unexpectedly displayed lowpromoter potency on both sides. The bidirectional rCMV-CAG promoterdisplays bidirectional promoter potency that is lower from both sidescompared to the potency of the hCMV unidirectional control and alsolower on both sides and not as balanced compared to the bidirectionalhCMV-rhCMV promoter (of which data are shown in FIG. 2A). In a thirdscreening experiment two new bidirectional promoters rhCMV-CAG2, rCMVbidir.1, and the two already tested bidirectional promoters hCMV-rhCMVand hCMV-CAG4 were tested. While rhCMV-CAG2 showed bidirectionalpromoter potency albeit weaker compared to the unidirectional hCMVcontrol, rCMV bidir 1 only induced expression of eGFP placed on theright side of the promoter and Luciferase activity was in the range ofthe untransfected cells control. In this experiment 3 the bidirectionalpromoters hCMV-rhCMV and hCMV-CAG4 were tested again confirming theirpromising potency and balance. As expected in a such a cell basedbiological transfection experiment, some variation is observed betweenexperiment 1 and experiment 3. Both hCMV-rhCMV and hCMV-CAG4 show lowerpotency in experiment 3 than in experiment 1, compared to theunidirectional hCMV promoter control. While hCMV-rhCMV is less potentthan hCMV-CAG4, it is more balanced with respect to eGFP and Luciferaseexpression. Thus, hCMV-rhCMV is a potent bidirectional promoter withvery balanced transgene expression, and also has the advantage of beingrelatively short.

Surprisingly, not all combinations of building blocks resulted in potentand balanced bidirectional promoters. For example hCMV-CAG4 andrhCMV-CAG1 and rhCMV-CAG2 are similar in terms of their building blocks.The three different designs all make use of a described strong CMVderived enhancer and promoter, albeit from different species, and thesame previously described strong CAG promoter. However, surprisinglyhCMV-CAG4 is more potent than rhCMV-CAG1 and rhCMV-CAG2. Additionallythe CAG promoter part was described to be stronger than an hCMVpromoter, however in the bidirectional setting, expression of thetransgene coupled to the hCMV promoter building block exceededexpression of the transgene coupled to the CAG promoter building block.This clearly demonstrates the unpredictability of creating newbidirectional promoters from previously known building blocks when usedin other constellations.

From the set of tested bidirectional promoter constructs hCMV-rhCMV wasidentified as the most balanced candidate of the potent bidirectionalpromoter constructs. From the design of the hCMV-rhCMV promoter frombuilding blocks of described potent promoter and enhancers, it could notbe predicted that this promoter would be both potent (although somewhatless potent than the other novel hCMV-CAG4 bidirectional promoter) andvery balanced (much more balanced than the mCMV bidirectional promoterthat was described in WO 2016/166088, and even somewhat more balancedthan the hCMV-CAG4 bidirectional promoter). Additionally, thebidirectional hCMV-rhCMV promoter has the advantage of beingconsiderably shorter (having a length of below 1 kB) than the otherpromoter designs. This means that use of this bidirectional promoterleaves more space for transgenes (i.e. allows longer transgenes) invectors that have a space limitation, such as rAds, compared to theother bidirectional promoters. The remaining tested syntheticbidirectional promoter designs consisting of different unidirectionalpromoter building blocks mainly displayed good bidirectional promoterfunctions, however they were generally less potent and less balancedthan the hCMV-rhCMV promoter.

Comparable to the mouse CMV bidirectional promoter, the rCMV immediateearly promoter can be designed as a bidirectional promoter which ishowever less potent. The results show that it is unpredictable whichcombination of building blocks provides good functionality of abidirectional promoter (potent expression in both directions, i.e. atleast 10%, preferably at least 20%, more preferably at least 30% ofexpression under control of the hCMV unidirectional promoter).

A schematic representation of hCMV-rhCMV including annotations for theidentity and orientation of the building blocks is displayed in FIG. 3.The right side of the bidirectional hCMV-rhCMV promoter includes arhesus CMV promoter building block (rhCMV) and the left side of thebidirectional hCMV-rhCMV promoter includes the hCMV promoter buildingblock and the hCMV enhancer building block in an inverted orientation topoint to the left side of the bidirectional promoter in the samedirection as the hCMV promoter. While here the terms “left” and “right”are used for ease of description, the skilled person will immediatelyrecognize that the bidirectional hCMV-rhCMV promoter construct can alsobe turned around and used in the opposite orientation. It should also benoted that with its relatively small size of less than 1 kb, thehCMV-rhCMV promoter is well suited for use as a bidirectional promoterin a recombinant adenoviral vector.

Example 3: Potency and Balance of Transgene Expression from AdenoviralVectors Harbouring an hCMV-rhCMV Expression Cassette

To further asses potency and balance of expression from the E1 region ofadenoviral vectors, we generated Ad26 and Ad35 vectors harbouring ahCMV-rhCMV bidirectional expression cassette in the E1 region. Fourdifferent vectors, viz. Ad26. eGFP.hCMV-rhCMV.Luc,Ad26.Luc.hCMV-rhCMV.eGFP, Ad35.eGFP.hCMV-rhCMV.Luc andAd35.Luc.hCMV-rhCMV.eGFP, were generated to assess potency and balanceof reporter gene expression upon transduction of non-complementing A549cells. Transductions were performed at 100 VP/cell and 1000 VP/cell.Since results at both VP/cell ratios were similar, only results of the1000 VP/cell transductions are shown in FIG. 4. In order to estimate atenfold difference in expression 100 VP/cell and 1000 VP/cell,transductions of the positive control vectors Ad.Luc and Ad.eGFPexpressing the reporter genes under control of the unidirectional hCMVpromoter are shown. Panel 4A shows that hCMV-rhCMV induces potentexpression of both the reporter genes from an Ad26 E1 bidirectionalexpression cassette, with expression levels slightly lower than from theAd26.Luc and Ad26.eGFP control vectors at 1000 VP/cell.Ad26.Luc.hCMV-rhCMV.eGFP is further directly compared to Ad26.Luc.mCMVbidir.eGFP and shows reduced transgene expression of eGFP compared tomCMV bidir., however also shows an overall more balanced transgeneexpression. Panel 4B shows transgene expression from an hCMV-rhCMVbidirectional expression cassette in Ad35 vectors. Interestingly, theexpression profile in Ad35 vectors slightly differed from the expressionprofile in Ad26 vectors. Therefore, while potent bidirectional promoterscan be used in rAdV derived from different serotypes, a differentpromoter may be optimal for use in one rAdV over another, furtherexemplifying that intricate design of promoters and expression cassettesis required for optimal viral vectors.

Example 4: Genetic Stability Testing of Adenoviral Vectors Harboring anhCMV-rhCMV

Bidirectional Expression Cassette in E1 Region 00128 Besides transgeneexpression, genetic stability during the production of AdV is a crucialparameter for a useful AdV expressing two antigens. Therefore geneticstability was tested as described in a previous application, WO2016/166088. Briefly, the vectors Ad26.Luc.hCMV-rhCMV.eGFP andAd26.eGFP.hCMV-rhCMV.Luc were generated by plasmid transfection inPER.C6 cells and viral populations were isolated by plaque picking. Tenplaques per vector were propagated to viral passage number (vpn) 3. Fromthere on, five plaques were selected for extended passaging up to vpn13. Genetic stability was evaluated by identity PCRs on the E1expression cassette region (FIG. 5), and E3 and E4 region (data of E3and E4 PCRs are not shown). Absence of small deletions or pointmutations was confirmed by standard Sanger sequencing of the E1 PCRproduct of vpn 13. Five out of five plaques of bothAd26.Luc.hCMV-rhCMV.eGFP and Ad26.eGFP.hCMV-rhCMV.Luc remainedgenetically stable up to vpn 13.

CONCLUSION

As described supra, by screening a panel of new bidirectional promoterconstructs, it was determined that it is unpredictable whichbidirectional promoter constructs will give the desired promoterproperties. In fact, even bidirectional promoter constructs that seem tobe very similar do not necessarily give the same results. For example,the bidirectional hCMV-rhCMV promoter, with a human CMV promoter (hCMV)on the left side and a short rhesus CMV promoter (rhCMV) on the rightside, showed particularly balanced expression of two differenttransgenes from the E1 region of rAd26 and rAd35 vectors. Surprisinglythe bidirectional hCMV-rhCMV promoter combined potency and balance oftransgene expression, and is also quite small with a length of below 1kB. rAd with the bidirectional hCMV-rhCMV promoter were determined to begenetically stable even after serial passaging in PER.C6 cells to P13.Thus, unpredictably, the bidirectional hCMV-rhCMV promoter of thepresent invention is a promoter with surprisingly preferablecharacteristics for use in recombinant viral vectors that can be used ingene therapy or vaccines where particularly balanced and potentexpression are desired and/or where the small size of the bidirectionalhCMV-rhCMV promoter is useful.

TABLE 1 Potency Potency right left side side (compared (compared to toGenetic Bidirectional standard) standard) stability in promoter SizeBalance in % in % AdV hCMV- 943 ~1.3/1-1.8/1 ~30 ~30 confirmed rhCMVmCMV* 1958 ~1/10 ~100 ~1000 confirmed *described in WO 2016/166088

REFERENCES U.S. Patent Documents

-   U.S. Pat. No. 5,057,540A (Oct. 15, 1991). “Saponin adjuvant”.    Kensil, Charlotte A.; Marciani, Dante J.-   U.S. Pat. No. 5,122,458A (Jun. 16, 1992). “Use of a bGH gDNA    polyadenylation signal in expression of non-bGH polypeptides in    higher eukaryotic cells”. Post, Leonard E.; Palermo, Daniel P.;    Thomsen, Darrell R.; Rottman, Fritz M.; Goodwin, Edward C.; Woychik,    Richard P.-   U.S. Pat. No. 5,559,099A (Sep. 24, 1996). “Penton base protein and    methods of using same”. Wickham, Thomas J.; Kovesdi, Imre; Brough,    Douglas E.; McVey, Duncan L.; Brader, Joseph T.-   U.S. Pat. No. 5,837,511A (Nov. 17, 1998). “Non-group C adenoviral    vectors”. Falck Pedersen, Erik S.; Crystal, Ronald G.; Mastrangeli,    Andrea; Abrahamson, Karil-   U.S. Pat. No. 5,837,520A (Nov. 17, 1998). “Method of purification of    viral vectors”. Shabram, Paul W.; Huyghe, Bernard G.; Liu, Xiaodong;    Shepard, H. Michael-   U.S. Pat. No. 5,846,782A (Dec. 8, 1998). “Targeting adenovirus with    use of constrained peptide motifs”. Wickham, Thomas J.; Roelvink,    Petrus W.; Kovesdi, Imre-   U.S. Pat. No. 5,851,806A (Dec. 22, 1998). “Complementary adenoviral    systems and cell lines”. Kovesdi, Imre; Brough, Douglas E.; McVey,    Duncan L.; Bruder, Joseph T.; Lizonova, Alena-   U.S. Pat. No. 5,891,690A (Apr. 6, 1999). “Adenovirus    E1-complementing cell lines”. Massie, Bernard-   U.S. Pat. No. 5,965,541A (Oct. 12, 1999). “Vectors and methods for    gene transfer to cells”. Wickham, Thomas J.; Kovesdi, Imre; Brough,    Douglas E.-   U.S. Pat. No. 5,981,225A (Nov. 9, 1999). “Gene transfer vector,    recombinant adenovirus particles containing the same, method for    producing the same and method of use of the same”. Kochanek, Stefan;    Schiedner, Gudrun-   U.S. Pat. No. 5,994,106A (Nov. 30, 1999). “Stocks of recombinant,    replication-deficient adenovirus free of replication-competent    adenovirus”. Kovesdi, Imre; Brough, Douglas E.; McVey, Duncan L.;    Bruder, Joseph T.; Lizonova, Alena-   U.S. Pat. No. 5,994,128A (Nov. 30, 1999). “Packaging systems for    human recombinant adenovirus to be used in gene therapy”. Fallaux,    Frits Jacobus; Hoeben, Robert Cornelis; Van der Eb, Alex Jan; Bout,    Abraham; Valerio, Domenico-   U.S. Pat. No. 6,020,191A (Feb. 1, 2000). “Adenoviral vectors capable    of facilitating increased persistence of transgene expression”.    Scaria, Abraham; Gregory, Richard J.; Wadsworth, Samuel C.-   U.S. Pat. No. 6,040,174A (Mar. 21, 2000). “Defective adenoviruses    and corresponding complementation lines”. Imler, Jean Luc; Mehtali,    Majid; Pavirani, Andrea-   U.S. Pat. No. 6,083,716A (Jul. 4, 2000). “Chimpanzee adenovirus    vectors”. Wilson, James M.; Farina, Steven F.; Fisher, Krishna J.-   U.S. Pat. No. 6,113,913A (Sep. 5, 2000). “Recombinant adenovirus”.    Brough, Douglas E.; Kovesdi, Imre-   U.S. Pat. No. 6,225,289B1 (May 1, 2001). “Methods and compositions    for preserving adenoviral vectors”. Kovesdi, Imre; Ransom, Stephen    C.-   U.S. Pat. No. 6,261,823B1 (Jul. 17, 2001). “Methods for purifying    viruses”. Tang, John Chu Tay; Vellekamp, Gary; Bondoc, Jr., Laureano    L.-   U.S. Pat. No. 6,485,958B2 (Nov. 26, 2002). “Method for producing    recombinant adenovirus”. Blanche, Francis; Guillaume, Jean Marc-   U.S. Pat. No. 7,326,555B2 (Feb. 5, 2008). “Methods of adenovirus    purification”. Konz, Jr., John O.; Lee, Ann L.; To, Chi Shung Brian;    Goerke, Aaron R-   U.S. Pat. No. 8,932,607B2 (Jan. 13, 2015). “Batches of recombinant    adenovirus with altered terminal ends”. Custers, Jerome H. H. V.;    Vellinga, Jort

European Patent Documents

-   EP1230354B1 (Jan. 7, 2004). “PERMANENT AMNIOCYTE CELL LINE, THE    PRODUCTION THEREOF AND ITS USE FOR PRODUCING GENE TRANSFER VECTORS”.    KOCHANEK, Stefan; SCHIEDNER, Gudrun-   EP1601776B1 (Jul. 2, 2008). “EXPRESSION VECTORS COMPRISING THE MCMV    1E2 PROMOTER”. CHATELLARD, Philippe; IMHOF, Markus-   EP853660B1 (Jan. 22, 2003). “METHOD FOR PRESERVING INFECTIOUS    RECOMBINANT VIRUSES, AQUEOUS VIRAL SUSPENSION AND USE AS MEDICINE”.    SENE, Claude

International Patent Application Publications

-   WO2003049763A1 (Jun. 19, 2003). “COMPOSITION FOR THE PRESERVATION OF    VIRUSES”. SETIAWAN, Kerrie; CAMERON, Fiona, Helen-   WO2003061708A1 (Jul. 31, 2003). “STABILIZED FORMULATIONS OF    ADENOVIRUS”. PUNGOR, Erno-   WO2003078592A2 (Sep. 25, 2003). “METHOD FOR THE PURIFICATION,    PRODUCTION AND FORMULATION OF ONCOLYTIC ADENOVIRUSES”. MEMARZADEH,    Bahram; PENNATHUR-DAS, Rukmini; WYPYCH, Joseph; YU, De Chao-   WO2003104467A1 (Dec. 18, 2003). “MEANS AND METHODS FOR THE    PRODUCTION OF ADENOVIRUS VECTORS”. VOGELS, Ronald; BOUT, Abraham-   WO2004001032A2 (Dec. 31, 2003). “STABLE ADENOVIRAL VECTORS AND    METHODS FOR PROPAGATION THEREOF”. VOGELS, Ronald; HAVENGA, Menzo,    Jans, Emco; ZUIJDGEEST, David, Adrianus, Theodorus-   WO2004004762A1 (Jan. 15, 2004). “ISCOM PREPARATION AND USE THEREOF”.    MOREIN, Bror; LOVGREN BENGTSSON, Karin-   WO2004020971A2 (Mar. 11, 2004). “CHROMATOGRAPHIC METHODS FOR    ADENOVIRUS PURIFICATION”. SENESAC, Joseph-   WO2004037294A2 (May 6, 2004). “NEW SETTINGS FOR RECOMBINANT    ADENOVIRAL-BASED VACCINES”. HAVENGA, Menzo, Jans, Emco; HOLTERMAN,    Lennart; KOSTENSE, Stefan; PAU, Maria, Grazia; SPRANGERS, Mieke,    Caroline; VOGELS, Ronald-   WO2004055187A1 (Jul. 1, 2004). “RECOMBINANT VIRAL-BASED MALARIA    VACCINES”. PAU, Maria Grazia; HOLTERMAN, Lennart; KASPERS, Jorn;    STEGMANN, Antonius, Johannes, Hendrikus-   WO2005002620A1 (Jan. 13, 2005). “QUIL A FRACTION WITH LOW TOXICITY    AND USE THEREOF”. MOREIN, Bror; LOVGREN BENGTSSON, Karin; EKSTROM,    Jill; RANLUND, Katarina-   WO2005071093A2 (Aug. 4, 2005). “CHIMPANZEE ADENOVIRUS VACCINE    CARRIERS”. CIRILLO, Agostino; COLLOCA, Stefano; ERCOLE, Bruno,    Bruni; MEOLA, Annalisa; NICOSIA, Alfredo; SPORENO, Elisabetta-   WO2005080556A2 (Sep. 1, 2005). “VIRUS PURIFICATION METHODS”.    WEGGEMAN, Miranda; VAN CORVEN, Emile Joannes Josephus Maria-   WO2006053871A2 (May 26, 2006). “MULTIVALENT VACCINES COMPRISING    RECOMBINANT VIRAL VECTORS”. HAVENGA, Menzo, Jans, Emco; VOGELS,    Ronald; SADOFF, Jerald; HONE, David; SKFIKY, Yasir Abdul Wahid;    RADOSEVIC, Katarina-   WO2006108707A1 (Oct. 19, 2006). “VIRUS PURIFICATION USING    ULTRAFILTRATION”. WEGGEMAN, Miranda-   WO2006120034A1 (Nov. 16, 2006). “VACCINE COMPOSITION”. ERTL, Peter,    Franz; TITE, John, Philip; VAN WELY, Catherine Ann-   WO2007073513A2 (Jun. 28, 2007). “METHOD FOR PROPAGATING ADENOVIRAL    VECTORS ENCODING INHIBITORY GENE PRODUCTS”. GALL, Jason, G., D.;    BROUGH, Douglas, E.; RICHTER, King, C.-   WO2007100908A2 (Sep. 7, 2007). “CHIMERIC ADENOVIRAL VECTORS”.    TUCKFR, Sean, N.-   WO2007104792A2 (Sep. 20, 2007). “RECOMBINANT ADENOVIRUSES BASED ON    SEROTYPE 26 AND 48, AND USE THEREOF”. BAROUCH, Dan H.; HAVENGA,    Menzo Jans Emko-   WO2007110409A1 (Oct. 4, 2007). “COMPOSITIONS COMPRISING A    RECOMBINANT ADENOVIRUS AND AN ADJUVANT”. HAVENGA, Menzo Jans Emko;    RADOSEVIC, Katarina-   WO2009026183A1 (Feb. 26, 2009). “USE OF CHIMERIC HIV/SIV GAG    PROTEINS TO OPTIMIZE VACCINE-INDUCED T CELL RESPONSES AGAINST HIV    GAG”. NABEL, Gary, J.; YANG, Zhi-Yong; SHI, Wei; BAROUCH, Dan, H.-   WO2009117134A2 (Sep. 24, 2009). “AEROSOLIZED GENETIC VACCINES AND    METHODS OF USE”. ROEDERER, Mario; RAO, Srinivas; NABEL, Gary, J.;    ANDREWS, Charla, Anne-   WO2010085984A1 (Aug. 5, 2010). “SIMIAN ADENOVIRUS NUCLEIC ACID- AND    AMINO ACID-SEQUENCES, VECTORS CONTAINING SAME, AND USES THEREOF”.    COLLOCA, Stefano; NICOSIA, Alfredo; CORTESE, Riccardo; AMMENDOLA,    Virginia; AMBROSIO, Maria-   WO2010086189A2 (Aug. 5, 2010). “SIMIAN ADENOVIRUS NUCLEIC ACID- AND    AMINO ACID-SEQUENCES, VECTORS CONTAINING SAME, AND USES THEREOF”.    COLLOCA, Stefano; NICOSIA, Alfredo; CORTESE, Riccardo; AMMENDOLA,    Virginia; AMBROSIO, Maria-   WO2010096561A1 (Aug. 26, 2010). “SYNTHETIC HIV/SIV GAG PROTEINS AND    USES THEREOF”. NABEL, Gary J.; YANG, Zhi-yong; SHI, Wei; BAROUCH,    Dan H.-   WO2011045378A1 (Apr. 21, 2011). “METHOD FOR THE PURIFICATION OF    ADENOVIRUS PARTICLES”. DE VOCHT, Marcel, Leo; VEENSTRA, Marloes-   WO2011045381A1 (Apr. 21, 2011). “PROCESS FOR ADENOVIRUS PURIFICATION    FROM HIGH CELL DENSITY CULTURES”. DE VOCHT, Marcel, Leo; VEENSTRA,    Marloes-   WO2013139911A1 (Sep. 26, 2013). “VACCINE AGAINST RSV”. RADOSEVIC,    Katarina; CUSTERS, Jerôme H. H. V.; VELLINGA, Jort; WIDJOJOATMODJO,    Myra N.-   WO2013139916A1 (Sep. 26, 2013). “VACCINE AGAINST RSV”. RADOSEVIC,    Katarina; CUSTERS, Jerôme H. H. V.; VELLINGA, Jort; WIDJOJOATMODJO,    Myra, N.

OTHER REFERENCES Books

-   Ausubel et al., Current Protocols in Molecular Biology, Wiley    Interscience Publishers, NY (1995)-   Ausubel F. M., et al. (editors). Current Protocols in Molecular    Biology; the series Methods in Enzymology, Academic Press, Inc.    (1987)-   Freshney, R. I., Culture of animal cells: A manual of basic    technique, fourth edition, Wiley-Liss Inc., ISBN 0-471-34889-9    (2000)-   Frokjaer S. and Hovgaard L. (editors), Pharmaceutical Formulation    Development of Peptides and Proteins, Taylor & Francis (2000)-   Gennaro, A. R. (editor), Remington's Pharmaceutical Sciences, 18th    edition, Mack Publishing Company (1990)-   Horowitz, M. S., Adenoviruses, Chapter 68, in Virology, (B. N.    Fields et al. (editors), 3rd Ed., Raven Press, Ltd., New York (1996)-   Kibbe A. (editor), Handbook of Pharmaceutical Excipients, 3rd    edition, Pharmaceutical Press (2000)-   Kruse and Paterson (editors), Tissue Culture, Academic Press. (1973)-   MacPherson M. J., Hams B. D., Taylor G. R. (editors), PCR2: A    Practical Approach (1995)-   Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd Ed.,    Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)-   Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory    Manual, 2nd Ed., (1989)-   Shenk, Thomas, Adenoviridae and their Replication, Chapter 67, in    Virology, B. N. Fields et al. (editors)., 3rd Ed., Raven Press,    Ltd., New York (1996)-   Watson et al., Recombinant DNA, 2nd ed., Scientific American Books.    (1992)

Journals

-   Abbink, P., Lemckert, A. A., Ewald, B. A., Lynch, D. M., Denholtz,    M., Smits, S., . . . Barouch, D. H. (2007). Comparative    seroprevalence and immunogenicity of six rare serotype recombinant    adenovirus vaccine vectors from subgroups B and D. J Virol, 81(9),    4654-4663. doi: 10.1128/JVI.02696-06-   Abbink, P., Maxfield, L. F., Ng'ang'a, D., Borducchi, E. N.,    Iampietro, M. J., Bricault, C. A., . . . Barouch, D. H. (2015).    Construction and evaluation of novel rhesus monkey adenovirus    vaccine vectors. J Virol, 89(3), 1512-1522. doi:    10.1128/JVI.02950-14-   Abrahamsen, K., Kong, H. L., Mastrangeli, A., Brough, D., Lizonova,    A., Crystal, R. G., & Falck-Pedersen, E. (1997). Construction of an    adenovirus type 7a E1A-vector. J Virol, 71(11), 8946-8951.-   Addison, C. L., Hitt, M., Kunsken, D., & Graham, F. L. (1997).    Comparison of the human versus murine cytomegalovirus immediate    early gene promoters for transgene expression by adenoviral vectors.    J Gen Virol, 78 (Pt 7), 1653-1661. doi: 10.1099/0022-1317-78-7-1653-   Amendola, M., Venneri, M. A., Biffi, A., Vigna, E., & Naldini, L.    (2005). Coordinate dual-gene transgenesis by lentiviral vectors    carrying synthetic bidirectional promoters. Nat Biotechnol, 23(1),    108-116.-   Andrianaki, A., Siapati, E. K., Hirata, R. K., Russell, D. W., &    Vassilopoulos, G. (2010). Dual transgene expression by foamy virus    vectors carrying an endogenous bidirectional promoter. Gene Ther,    17(3), 380-388. doi: 10.1038/gt.2009.147-   Bangari, D. S., & Mittal, S. K. (2006). Development of nonhuman    adenoviruses as vaccine vectors. Vaccine, 24(7), 849-862. doi:    10.1016/j.vaccine.2005.08.101-   Barry, P. A., Alcendor, D. J., Power, M. D., Kerr, H., &    Luciw, P. A. (1996). Nucleotide sequence and molecular analysis of    the rhesus cytomegalovirus immediate-early gene and the UL121-117    open reading frames. Virology, 215(1), 61-72. doi:    10.1006/viro.1996.0007-   Barski, O. A., Siller-Lopez, F., Bohren, K. M., Gabbay, K. H., &    Aguilar-Cordova, E. (2004). Human aldehyde reductase promoter allows    simultaneous expression of two genes in opposite directions.    Biotechniques, 36(3), 382-384, 386, 388.-   Belousova, N., Harris, R., Zinn, K., Rhodes-Selser, M. A., Kotov,    A., Kotova, O., . . . Alvarez, R. D. (2006). Circumventing    recombination events encountered with production of a clinical-grade    adenoviral vector with a double-expression cassette. Mol Pharmacol,    70(5), 1488-1493.-   Brough, D. E., Lizonova, A., Hsu, C., Kulesa, V. A., & Kovesdi, I.    (1996). A gene transfer vector-cell line system for complete    functional complementation of adenovirus early regions E1 and E4. J    Virol, 70(9), 6497-6501.-   Chan, Y. J., Chiou, C. J., Huang, Q., & Hayward, G. S. (1996).    Synergistic interactions between overlapping binding sites for the    serum response factor and ELK-1 proteins mediate both basal    enhancement and phorbol ester responsiveness of primate    cytomegalovirus major immediate-early promoters in monocyte and    T-lymphocyte cell types. J Virol, 70(12), 8590-8605.-   Chang, Y. N., Jeang, K. T., Chiou, C. J., Chan, Y. J., Pizzorno, M.,    & Hayward, G. S. (1993). Identification of a large bent DNA domain    and binding sites for serum response factor adjacent to the NFI    repeat cluster and enhancer region in the major IE94 promoter from    simian cytomegalovirus. J Virol, 67(1), 516-529.-   Chatellard, P., Pankiewicz, R., Meier, E., Durrer, L., Sauvage, C.,    & Imhof, M. O. (2007). The IE2 promoter/enhancer region from mouse    CMV provides high levels of therapeutic protein expression in    mammalian cells. Biotechnol Bioeng, 96(1), 106-117. doi:    10.1002/bit.21172-   Cohen, C. J., Xiang, Z. Q., Gao, G. P., Ertl, H. C., Wilson, J. M.,    & Bergelson, J. M. (2002). Chimpanzee adenovirus CV-68 adapted as a    gene delivery vector interacts with the coxsackievirus and    adenovirus receptor. J Gen Virol, 83(Pt 1), 151-155.-   Collins, P. J., Kobayashi, Y., Nguyen, L., Trinklein, N. D., &    Myers, R. M. (2007). The ets-related transcription factor GABP    directs bidirectional transcription. PLoS Genet, 3(11), e208. doi:    10.1371/journal.pgen.0030208-   Fallaux, F. J., Bout, A., van der Velde, I., van den Wollenberg, D.    J., Hehir, K. M., Keegan, J., . . . Hoeben, R. C. (1998). New helper    cells and matched early region 1-deleted adenovirus vectors prevent    generation of replication-competent adenoviruses. Hum Gene Ther,    9(13), 1909-1917.-   Farina, S. F., Gao, G. P., Xiang, Z. Q., Rux, J. J., Burnett, R. M.,    Alvira, M. R., . . . Wilson, J. M. (2001). Replication-defective    vector based on a chimpanzee adenovirus. J Virol, 75(23),    11603-11613. doi: 10.1128/JVI.75.23.11603-11613.2001-   Gao, G. P., Engdahl, R. K., & Wilson, J. M. (2000). A cell line for    high-yield production of E1-deleted adenovirus vectors without the    emergence of replication-competent virus. Hum Gene Ther, 11(1),    213-219. doi: 10.1089/10430340050016283-   Geisbert, T. W., Bailey, M., Hensley, L., Asiedu, C., Geisbert, J.,    Stanley, D., . . . Sullivan, N. J. (2011). Recombinant adenovirus    serotype 26 (Ad26) and Ad35 vaccine vectors bypass immunity to Ad5    and protect nonhuman primates against ebolavirus challenge. J Virol,    85(9), 4222-4233. doi: 10.1128/JVI.02407-10-   Goerke, A. R., To, B. C., Lee, A. L., Sagar, S. L., & Konz, J. O.    (2005). Development of a novel adenovirus purification process    utilizing selective precipitation of cellular DNA. Biotechnol    Bioeng, 91(1), 12-21. doi: 10.1002/bit.20406-   Hansen, S. G., Strelow, L. I., Franchi, D. C., Anders, D. G., &    Wong, S. W. (2003). Complete sequence and genomic analysis of rhesus    cytomegalovirus. J Virol, 77(12), 6620-6636.-   Harro, C. D., Robertson, M. N., Lally, M. A., O'Neill, L. D.,    Edupuganti, S., Goepfert, P. A., . . . Mehrotra, D. V. (2009).    Safety and immunogenicity of adenovirus-vectored near-consensus HIV    type 1 Glade B gag vaccines in healthy adults. AIDS Res Hum    Retroviruses, 25(1), 103-114.-   Harro, C., Sun, X., Stek, J. E., Leavitt, R. Y., Mehrotra, D. V.,    Wang, F., . . . Merck, V. Study Group. (2009). Safety and    immunogenicity of the Merck adenovirus serotype 5 (MRKAd5) and    MRKAd6 human immunodeficiency virus type 1 trigene vaccines alone    and in combination in healthy adults. Clin Vaccine Immunol, 16(9),    1285-1292. doi: 10.1128/CVI.00144-09-   Havenga, M., Vogels, R., Zuijdgeest, D., Radosevic, K., Mueller, S.,    Sieuwerts, M., . . . Goudsmit, J. (2006). Novel    replication-incompetent adenoviral B-group vectors: high vector    stability and yield in PER.C6 cells. J Gen Virol, 87(Pt 8),    2135-2143.-   Heilbronn, R., & Weger, S. (2010). Viral vectors for gene transfer:    current status of gene therapeutics. Handb Exp Pharmacol (197),    143-170. doi: 10.1007/978-3-642-00477-3_5-   Hoganson, D. K., Ma, J. C., Asato, L., Ong, M., Printz, M. A.,    Huyghe, B. G., . . . D'Andrea, M. J. (2002). Development of a Stable    Adenoviral Vector Formulation. BioProcessing J., 1(1), 43-48.-   Holman, D. H., Wang, D., Raviprakash, K., Raja, N. U., Luo, M.,    Zhang, J., . . . Dong, J. Y. (2007). Two complex, adenovirus-based    vaccines that together induce immune responses to all four dengue    virus serotypes. Clin Vaccine Immunol, 14(2), 182-189.-   Holterman, L., Vogels, R., van der Vlugt, R., Sieuwerts, M.,    Grimbergen, J., Kaspers, J., . . . Havenga, M. (2004). Novel    replication-incompetent vector derived from adenovirus type 11    (Ad11) for vaccination and gene therapy: low seroprevalence and    non-cross-reactivity with Ad5. J Virol, 78(23), 13207-13215. doi:    10.1128/JVI.78.23.13207-13215.2004-   Hu, X., Meng, W., Dong, Z., Pan, W., Sun, C., & Chen, L. (2011).    Comparative immunogenicity of recombinant adenovirus-vectored    vaccines expressing different forms of hemagglutinin (HA) proteins    from the H5 serotype of influenza A viruses in mice. Virus Res,    155(1), 156-162. doi: 10.1016/j.virusres.2010.09.014-   Kim, D. W., Uetsuki, T., Kaziro, Y., Yamaguchi, N., & Sugano, S.    (1990). Use of the human elongation factor 1 alpha promoter as a    versatile and efficient expression system. Gene, 91(2), 217-223.-   Kobinger, G. P., Feldmann, H., Zhi, Y., Schumer, G., Gao, G.,    Feldmann, F., . . . Wilson, J. M. (2006). Chimpanzee adenovirus    vaccine protects against Zaire Ebola virus. Virology, 346(2),    394-401. doi: 10.1016/j.virol.2005.10.042-   Lasaro, M. O., & Ertl, H. C. (2009). New insights on adenovirus as    vaccine vectors. Mol Ther, 17(8), 1333-1339. doi:    10.1038/mt.2009.130-   Lemckert, A. A., Grimbergen, J., Smits, S., Hartkoorn, E.,    Holterman, L., Berkhout, B., . . . Havenga, M. J. (2006). Generation    of a novel replication-incompetent adenoviral vector derived from    human adenovirus type 49: manufacture on PER.C6 cells, tropism and    immunogenicity. J Gen Virol, 87(Pt 10), 2891-2899. doi:    10.1099/vir.0.82079-0-   Mullick, A., Xu, Y., Warren, R., Koutroumanis, M., Guilbault, C.,    Broussau, S., . . . Massie, B. (2006). The cumate gene-switch: a    system for regulated expression in mammalian cells. BMC Biotechnol,    6, 43. doi: 10.1186/1472-6750-6-43-   Na, M., & Fan, X. (2010). Design of Ad5F35 vectors for coordinated    dual gene expression in candidate human hematopoietic stem cells.    Exp Hematol, 38(6), 446-452. doi: 10.1016/j.exphem.2010.03.007-   Nan, X., Peng, B., Hahn, T. W., Richardson, E., Lizonova, A.,    Kovesdi, I., & Robert-Guroff, M. (2003). Development of an Ad7    cosmid system and generation of an Ad7deltaE1deltaE3HIV(MN) env/rev    recombinant virus. Gene Ther, 10(4), 326-336. doi:    10.1038/sj.gt.3301903-   Ogun, S. A., Dumon-Seignovert, L., Marchand, J. B., Holder, A. A., &    Hill, F. (2008). The oligomerization domain of C4-binding protein    (C4 bp) acts as an adjuvant, and the fusion protein comprised of the    19-kilodalton merozoite surface protein 1 fused with the murine C4    bp domain protects mice against malaria. Infect Immun, 76(8),    3817-3823. doi: 10.1128/IAI.01369-07-   Ophorst, O. J., Radosevic, K., Havenga, M. J., Pau, M. G.,    Holterman, L., Berkhout, B., . . . Tsuji, M. (2006). Immunogenicity    and protection of a recombinant human adenovirus serotype 35-based    malaria vaccine against Plasmodium yoelii in mice. Infect Immun,    74(1), 313-320.-   Pham, L., Nakamura, T., Gabriela Rosales, A., Carlson, S. K.,    Bailey, K. R., Peng, K. W., & Russell, S. J. (2009). Concordant    activity of transgene expression cassettes inserted into E1, E3 and    E4 cloning sites in the adenovirus genome. J Gene Med, 11(3),    197-206.-   Post, D. E., & Van Meir, E. G. (2001). Generation of bidirectional    hypoxia/HIF-responsive expression vectors to target gene expression    to hypoxic cells. Gene Ther, 8(23), 1801-1807. doi:    10.1038/sj.gt.3301605-   Powell, S. K., Rivera-Soto, R., & Gray, S. J. (2015). Viral    expression cassette elements to enhance transgene target specificity    and expression in gene therapy. Discov Med, 19(102), 49-57.-   Richardson, J. S., Yao, M. K., Tran, K. N., Croyle, M. A.,    Strong, J. E., Feldmann, H., & Kobinger, G. P. (2009). Enhanced    protection against Ebola virus mediated by an improved    adenovirus-based vaccine. PLoS One, 4(4), e5308. doi:    10.1371/journal.pone.0005308-   Robbins, P. D., & Ghivizzani, S. C. (1998). Viral vectors for gene    therapy. Pharmacol Ther, 80(1), 35-47.-   Sandford, G. R., & Burns, W. H. (1996). Rat cytomegalovirus has a    unique immediate early gene enhancer. Virology, 222(2), 310-317.    doi: 10.1006/viro.1996.0428-   Schepp-Berglind, J., Luo, M., Wang, D., Wicker, J. A., Raja, N. U.,    Hoel, B. D., . . . Dong, J. Y. (2007). Complex adenovirus-mediated    expression of West Nile virus C, PreM, E, and NS1 proteins induces    both humoral and cellular immune responses. Clin Vaccine Immunol,    14(9), 1117-1126.-   Sullivan, N. J., Geisbert, T. W., Geisbert, J. B., Shedlock, D. J.,    Xu, L., Lamoreaux, L., . . . Nabel, G. J. (2006). Immune protection    of nonhuman primates against Ebola virus with single low-dose    adenovirus vectors encoding modified GPs. PLoS Med, 3(6), e177. doi:    10.1371/journal.pmed.0030177-   Sullivan, N. J., Geisbert, T. W., Geisbert, J. B., Xu, L., Yang, Z.    Y., Roederer, M., . . . Nabel, G. J. (2003). Accelerated vaccination    for Ebola virus haemorrhagic fever in non-human primates. Nature,    424(6949), 681-684. doi: 10.1038/nature01876-   Tatsis, N., Blejer, A., Lasaro, M. O., Hensley, S. E., Cun, A.,    Tesema, L., . . . Ertl, H. C. (2007). A CD46-binding chimpanzee    adenovirus vector as a vaccine carrier. Mol Ther, 15(3), 608-617.    doi: 10.1038/sj.mt.6300078-   Vemula, S. V., & Mittal, S. K. (2010). Production of adenovirus    vectors and their use as a delivery system for influenza vaccines.    Expert Opin Biol Ther, 10(10), 1469-1487. doi:    10.1517/14712598.2010.519332-   Vogels, R., Zuijdgeest, D., van Meerendonk, M., Companjen, A.,    Gillissen, G., Sijtsma, J., . . . Havenga, M. J. (2007). High-level    expression from two independent expression cassettes in    replication-incompetent adenovirus type 35 vector. J Gen Virol,    88(Pt 11), 2915-2924.-   Vogels, R., Zuijdgeest, D., van Rijnsoever, R., Hartkoorn, E.,    Damen, I., de Bethune, M. P., . . . Havenga, M. (2003).    Replication-deficient human adenovirus type 35 vectors for gene    transfer and vaccination: efficient human cell infection and bypass    of preexisting adenovirus immunity. J Virol, 77(15), 8263-8271.-   Voigt, S., Sandford, G. R., Hayward, G. S., & Burns, W. H. (2005).    The English strain of rat cytomegalovirus (CMV) contains a novel    captured CD200 (vOX2) gene and a spliced CC chemokine upstream from    the major immediate-early region: further evidence for a separate    evolutionary lineage from that of rat CMV Maastricht. J Gen Virol,    86(Pt 2), 263-274. doi: 10.1099/vir.0.80539-0-   Walther, W., & Stein, U. (2000). Viral vectors for gene transfer: a    review of their use in the treatment of human diseases. Drugs,    60(2), 249-271.-   Zhou, D., Cun, A., Li, Y., Xiang, Z., & Ertl, H. C. (2006). A    chimpanzee-origin adenovirus vector expressing the rabies virus    glycoprotein as an oral vaccine against inhalation infection with    rabies virus. Mol Ther, 14(5), 662-672. doi:    10.1016/j.ymthe.2006.03.027-   Zhou, D., Wu, T. L., Lasaro, M. O., Latimer, B. P., Parzych, E. M.,    Bian, A., . . . Ertl, H. C. (2010). A universal influenza A vaccine    based on adenovirus expressing matrix-2 ectodomain and nucleoprotein    protects mice from lethal challenge. Mol Ther, 18(12), 2182-2189.    doi: 10.1038/mt.2010.202

1. A recombinant nucleic acid molecule comprising a bidirectionalpromoter operably linked to a first transgene in one direction and to asecond transgene in the opposite direction, wherein the bidirectionalpromoter comprises a nucleotide sequence that is at least 90% identicalto SEQ ID NO:
 4. 2. The recombinant nucleic acid molecule of claim 1,wherein the bidirectional promoter comprises a nucleotide sequence thatis at least 95% identical to SEQ ID NO:
 4. 3. The recombinant nucleicacid molecule of claim 1, wherein the bidirectional promoter comprisesSEQ ID NO:
 4. 4. The recombinant nucleic acid molecule of claim 1,wherein the first and second transgene are different and at least one ofthem encodes an antigen.
 5. A recombinant vector or a recombinant virus,comprising a recombinant nucleic acid molecule according to claim
 1. 6.A method for expressing at least two transgenes in a cell, the methodcomprising providing a cell with a recombinant vector or a recombinantvirus according to claim
 5. 7. A method for inducing an immune responseagainst at least two antigens, the method comprising administering to asubject a recombinant vector or a recombinant virus according to claim5, wherein the first transgene encodes a first antigen and the secondtransgene encodes a second antigen different from the first antigen. 8.A pharmaceutical composition comprising a recombinant vector or arecombinant virus according to claim 5 and a pharmaceutically acceptablecarrier or excipient.
 9. A recombinant vector according to claim 5,wherein the vector is a plasmid vector.
 10. A recombinant virusaccording to claim 5, wherein the virus is an adenovirus.
 11. Therecombinant adenovirus of claim 10, wherein the adenovirus is a humanadenovirus serotype 35 or a human adenovirus serotype
 26. 12. Apharmaceutical composition comprising a recombinant virus according toclaim 10 and a pharmaceutically acceptable carrier or excipient.
 13. Arecombinant DNA molecule comprising the genome of a recombinantadenovirus, wherein said genome comprises a recombinant nucleic acidmolecule according to claim 1
 14. A recombinant DNA molecule comprisingthe genome of a recombinant adenovirus, wherein: (i) said genomecomprises a recombinant nucleic acid molecule according to claim 1; and(ii) the adenovirus has a deletion in the E1 region.
 15. A method ofproducing a genetically stable recombinant adenovirus comprising a firstand a second transgene that each are expressed when the adenovirusinfects a target cell, the method comprising: a) preparing a constructcomprising the recombinant nucleic acid molecule of claim 1; and b)incorporating said construct into the genome of the recombinantadenovirus.
 16. A method according to claim 15, wherein the first andsecond transgene are different and at least one of them encodes anantigen.
 17. A method according to claim 15, wherein the recombinantadenovirus is a human adenovirus serotype 35 or a human adenovirusserotype 26.